JPH11501520A - Nucleotide sequence of Haemophilus influenzae Rd genome, fragments thereof and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides the sequencing of the entire genome sequence of Haemophilus influenzae Rd, SEQ ID NO: 1. The invention further provides sequence information stored on a computer readable medium, and a computer-based system, and a method that facilitates its use. In addition to whole genome sequences, the present invention has identified over 1700 protein-encoding fragments of the genome and, by virtue of their location relative to the unique NotI restriction endonuclease site, Identify any modulatory elements that regulate expression.

Description

DETAILED DESCRIPTION OF THE INVENTION               The nucleotide sequence of the Rd genome of Haemophilus influenzae,                       The fragment and its use   Some of the work performed during the development of the present invention used US government funding. the government The present invention may have certain rights. NIH-5R01GM48251 Field of the invention   The present invention relates to the field of molecular biology. The present invention relates to H. influenzae ( Haemophilus influenzae) nucleotide sequence, including fragments thereof Disclosed are products and uses in industrial fermentation and pharmaceutical development. Background of the Invention   The complete genomic sequence of freely living cell organisms has not yet been determined. the first The Mycobacterium sequence was completed by 1996, while E. coli and S. cerevisiae were 19 It is expected to be completed by 98 years ago. These are overlap cosmid claw This is done by random and / or command sequencing. Random shotgun Attempts to determine sequences on the order of one megabase or more by the method No one was there.   Haemophilus influenzae is a small (approximately 0.4 × 1 Ron) Non-automatic, non-sporulating, embryo-negative bacteria. This is the upper part of children and adults Inhabits the respiratory mucosa and causes otitis media and respiratory tract infections in most children You. The most serious complication is meningitis, which is neurological in up to 50% of infected children Results in a clinical sequelae. Based on immunologically distinct capsular polysaccharide antigens, Fluenza serotypes (a to f) have been identified. Many strains whose type cannot be determined Is also known. Serotype b is responsible for the majority of human diseases.   Interest in the medically important features of Haemophilus influenzae biology is The focus is on the genes that determine the virulence characteristics of E. coli. Contributes to capsular polysaccharide Maps of a number of genes that have been generated and sequenced (Kroll et al., Mol . Microbiol.Five(6); 1549-1560 (1991). Some outer membrane proteins (OMP ) The gene was identified and sequenced (Langford et al., J. Gen. Microbi). l.138: 155-159 (1992)). Lipooligo bran (LOS) component of outer layer membrane and its combination Genes of the synthetic pathway have been intensively studied (Weiser et al., J. Bacteriol.172: 33 04-3309 (1990)). Although vaccines have been available since 1984, Studies have been motivated to some extent by the demand for improved vaccines. Recently Catalase gene is characterized as a potential virulence-related gene The sequence was determined (Bishni et al., Printing). Elucidation of H. influenzae genome How Influenza Bacteria Cause Invasive Diseases and How It will increase your understanding of how best to fight infection.   Haemophilus influenzae has a very efficient natural DNA transformation system and Leverage systems have been intensively studied with unencapsulated (R), serotype d strains (Kahn and Smith). J. Membrans Biology81: 89-103 (1984)). At least 16 transformants Alien genes have been identified and sequenced. Of these, four are regulated Gene (Redfield. J. Bacteriol.,173: 5612-5618 (1991) and Ch andler, Proc. Natl. Acad. Sci. USA89: 1626-1630 (1992)) At least two are involved in the recombination process (Barouki and Smith. J. Baoteriol. ,163(2): 629-634 (1985)), and at least seven toward membrane and periplasmic space (Tomb et al., Gene104: 1-10 (1991) and Tomb. Proc. Natl. A cad. Sci. USA89: 10252-10256 (1992)), and where these genes Seems to function as a structural component or within the assembly of the DNA transport machinery You. Transformation of Haemophilus influenzae Rd is based on front row specific DNA incorporation, Membrane, called the transformom, of several double-stranded DNA molecules per cell Rapid uptake into the chamber, linear translocation of single-stranded donor DNA into the cytoplasm Application and pairing and pairing of single strand and chromosome by single strand displacement mechanism Including replacement It shows a number of interesting features. Haemophilus influenzae Rd transformation system is gram shade The most thoroughly studied sexual system, and the Gram-positive system Is different.   The size of the Rd genome of Haemophilus influenzae is limited by a pulsed-field agaro It was determined to be approximately 1.9 Mb by gel electrophoresis, and this genome It is approximately 40% of the size of E. coli (Lee and Smith, J. Bacteriol.170: 4402 4054405 (1988)). The restriction map for Haemophilus influenzae is circular (Lee et al., J. Mol. Bacteriol.1713016-3024 (1989) and Redfield and Lee, "Influence Rd ", 2110-2112, O'Brien, SJ (ed.), Ganetic Maps: Loc. us Maps of Complexes Genomex, Cold Spring Harbor Press, New York H). Probes for Southern hybridization of various genes with restricted sinus DNA bands Mapped to restriction fragments by the method. This map is randomly distributed Demonstrates complete genomic sequence assembly from sequenced fragments It would be worth explaining. GenBank currently has about 100kb of redundancy There are no Haemophilus influenzae DNA sequences. About half are from serotype b And half from Rd                                Summary of the Invention   The invention is based on the sequencing of the Rd genome of Haemophilus influenzae. One obtained The following nucleotide sequence is provided in SEQ ID NO: 1.   The present invention relates to the obtained nucleotide sequence of the Rd genome or its substitutes. Forms in which tabular fragments can be easily used, analyzed and interpreted by skilled technicians To provide. In one embodiment, the present invention relates to a nucleic acid sequence represented by SEQ ID NO: 1. It is provided as a continuous sequence of primary sequence information corresponding to the otide sequence.   The invention further provides at least 99.9% identity to the nucleotide sequence of SEQ ID NO: 1. Provides the nucleotide sequence of   SEQ ID NO: 1 nucleotide sequence, a representative fragment or sequence identifier thereof A nucleotide sequence that is at least 99.9% identical to the nucleotide sequence of Alternative Number 1: Can be provided in a variety of media that facilitate the use of This embodiment In one such application, the sequences of the invention are described on a computer readable medium. . Such media include magnetic storage media such as floppy disks, hard disks, etc. Disk storage medium and magnetic chip; optical storage medium such as CD-ROM; RAM and Electrical storage media such as ROMs; and composites of these categories, for example magnetic / Optical storage media include, but are not limited to.   The invention further relates to a system, in particular a data storage means, stored and described herein. A computer-based system having sequence information provided. This The system integrates commercially important fragments of the Rd genome. It is designed to   Another embodiment of the present invention relates to an isolated fragment of the Rd genome of Haemophilus influenzae. Are turned to The fragments of the Rd genome of the Haemophilus influenzae of the present invention include , Fragment encoding peptide (hereinafter referred to as open reading frame (ORF)), operable A fragment that regulates the expression of an operatively linked ORF (hereinafter, expression regulation flag) (EMF)), a flag that mediates the uptake of bound DNA fragments into cells (Hereinafter referred to as uptake regulatory fragment (UMF)) and influenza in the sample. A fragment that can be used to diagnose the presence of Rd. (DF)), but is not limited thereto.   Each ORF fragment of the Haemophilus influenzae Rd genome is disclosed in Tables 1 (a) and 2 And the EMF found 5 'of the ORF is an enormous polynucleotide reagent. It can be used in a number of ways. These sequences indicate the presence of a particular microorganism in the sample. As a diagnostic probe or diagnostic amplification primer for Use to manufacture products and to selectively control gene expression Can be.   The invention further includes one or more of the Rd genomes of Haemophilus influenzae of the invention. And recombinant constructs containing the fragment. The recombinant construct of the present invention A vector into which a fragment of Haemophilus influenzae Rd is inserted, for example, Contains a sumid or viral vector.   The present invention further provides for the isolation of isolated fragments of the Rd genome of Haemophilus influenzae of the present invention. A host cell containing one of the following is provided. These host cells are Can be a higher eukaryotic host, such as a vesicle, or a lower eukaryotic cell, such as a yeast cell. Or prokaryotic cells such as bacterial cells.   The present invention is further directed to an isolated protein encoded by the ORF of the present invention. ing. The protein of the present invention may be prepared using various oils known in the art. Can be obtained. At the simplest level, it is commercially available Amino acid sequences can be synthesized using any suitable peptide synthesizer. Alternative In the method, the protein is purified from bacterial cells that naturally produce the protein. Most Later, the protein of the present invention has been modified to express the desired protein Alternatively, it can be purified from cells.   The present invention further provides a homologue of a fragment of Rd geno 4 of the Haemophilus influenzae of the present invention. And a method for obtaining a homolog of the protein encoded by the ORF of the present invention. provide. Specifically, the nucleotide and amino acid sequences disclosed herein are Use as lobes or primers and perform PCR cloning By using techniques such as Lark hybridization, Can obtain homologs.   The invention further provides an antibody that selectively binds to one of the proteins of the invention. . Such antibodies include both monoclonal and polyclonal antibodies. You.   The present invention further provides a hybridoma that produces the above antibody. Hybrid Ma is an immortalized cell line that can secrete specific monoclonal antibodies.   The present invention further relates to cells derived from cells expressing one of the ORFs of the present invention or homologs thereof. The present invention provides a method for identifying a test sample. Such a method uses a test sample of the present invention. Co-administered with one or more antibodies or one or more DFs of the invention Skilled in the art to determine whether the sample contains an ORF or a product produced therefrom. Incubating under conditions that can be measured by a person.   In another embodiment of the present invention, the reagents required to perform the above assay A kit is provided.   In particular, the present invention comprises (a) one of the antibodies of the present invention or one of the DFs A first container having; and (b) one or more of the following reagents: One or more reagents which can detect the presence of the antibody or hybridized DF. Close one or more containers, including more other containers, Provide a compartment kit for containment and acceptance.   Using the isolated protein of the present invention, the present invention further provides one of the ORFs of the present invention. Method for obtaining and identifying substances capable of binding to the encoded protein I will provide a. In particular, such substances include antibodies (described above), peptides, carbohydrates Goods, pharmaceuticals, etc. are included. Such a method involves the following steps:     (A) the substance is encoded by one of the ORFs of the invention              Contacting with the isolated protein; and     (B) Determine whether the substance binds to the protein              To do.   The complete genome sequence of Haemophilus influenzae is available for all studies It would be very valuable for the room and for a variety of commercial purposes. Haemophilus influenzae R d Many fragments of the genome can be stored in genebanks or protein databases. Immediately identified by similarity search, and for the time being Haemophilus researchers Valuable and immediate commercial for protein production or control of gene expression Worth it. A particular example concerns PHA synthase. Po Rehydroxybutyrate is present in the membrane of Haemophilus influenzae Rd and its Amounts have been reported to correlate with the level of competence required for transformation. this PHA synthase that synthesizes polymers has been identified and sequenced in a number of bacteria However, none of these bacteria are evolutionarily close to H. influenzae. this The gene is used in influenza using hybridization probes or PCR technology. It must still be isolated from the fungus. However, the genomic sequence of the present invention is as follows: The use of the described search means allows the identification of this gene.   The development of methodologies and technologies to elucidate the entire genome sequence of bacteria and other small genomes The ability and understanding to analyze chromosome tissue is greatly enhanced and enhanced Will be. In particular, the sequenced genome is located within a large fragment of genomic DNA. Ability to identify genes, structure, location and spacing of regulatory elements, potential for industrial application Identify genes with and perform comparative genomics and molecular phylogeny Provides models to develop tools for analyzing chromosome structure and function, including Will do.                                Description of the drawings   Figure 1-Restriction map of the Haemophilus influenzae Rd genome.   FIG. 2-Can be used to implement the computer-based system of the present invention. FIG.   Figure 3-2.5 Mb genome (triangle) with an average sequence length of 460 bp and 25 bp overlap. And 1.6Mb genome (circle) for Lander-Waterman Autoassembler (AtoAssembler) (square) compared to the prediction of n) Comparison of experimental ranges of up to approximately 4000 random sequence fragments.   Figure 4-Manage, assemble, edit and annotate the H. influenzae genome Data flow and computer program used to remove data. Macintosh A using both Macintosh and Unix platforms B373 sequence data file (Kerlavage et al., Proceedings of the Twenty-Sixt h Annual Hawaii International Conference on System Sciences, IE EE Computer Society Press, Washington DC, 585 (1993)) You. Factura (AB) for automatic vector sequence removal of sequence files And a Macintosh program designed for end trimming. Program Mu esp runs on the Macintosh platform and is Influenza based on Unix based feature data extracted from sequence files Write to the fungus-related database. Assembling, stp, X-window graphics Interface and user specified or standard SQL queries Control program that can be used to retrieve sequences from the Haemophilus influenzae database Identify using program Achieved by retrieving a set of sequence files and their related features . Sequence files are used to assemble thousands of sequence fragments quickly and accurately Assembled using TIGR assembler, TIGR designed assembly equipment Was set up. TIGR Editor (Editor) is TIGR assembler out Write an aligned sequence file from the put and use this alignment for continuous editing. Graphical interface capable of displaying columns and associated electropherograms Face. Identification of the putative coding region was determined by Genemark ( Borodovsky and McIninch, Computers Chem.17(2): 123 (1993)) Markov and Bayes modeling professionals to predict gene locations Gram and directed to a Haemophilus influenzae sequence dataset. peptide Search is Maspar MP-2 large with 4096 microprocessors Blaze (Brutag et al., Computers Chem) run on a large scale parallel computer .17: 203 (1993)) and 3 of each coding region predicted by Genemark Performed on one reading frame. The result obtained from each reading frame is calculated by mblzt. Combined in a single output file. Optimal protein alignment is latent Acquired using the program praze to extend the alignment between potential frameshifts Was. The output is an order that directly interacts with the Haemophilus influenzae database. -I checked it using gbyob, a graphic display program of Made. these Alignment is used to identify potential frameshift errors and further editing Goals and goals.   Figure 5-Location and geno location of each predicted coding region that has a match with the database Circular representation of the Rd chromosome of the Haemophilus influenzae showing selected general characteristics of the system . Outer circumference: unique NotI restriction site (designated nucleotide 1), RarII site And the location of the SmaI site. Outer concentric circle: For each identified coding region for which the gene was identified position. The position of each coding area is coded with respect to its role according to the color code in FIG. ing. Second concentric circle: high G / C content area (> 42%, red;> 40%, blue ) And regions with high A / T content (> 66%, black;> 64%, green). High G / C content The quantity region consists of six ribosomal operons and mu-like pro Of particular relevance to phages. Third concentric circle: range by lambda clone (blue). More than 300 lambda clones are sequenced from each end to provide a complete genome structure. Confirmed and identified six ribosomal operons. Fourth Concentric Circle: Six Revo Sosome operon (green), tRNA (black) and ambiguous mu-like prophage (blue) Color) position. Fifth concentric circle: simple line-up of repetitions. Indicates the position of the next iteration Are: CTGGCT, GTCT, ATT, AATGGC, TIGA, TT GG, TTTA, TTATC, TGAC, TCGTC, AACC, TTGG, C AAT, CCAA. Putative origin of replication is directed outwards starting around base 603,000 Indicated by the pointing arrow (green 6). The two potential termination sequences are on opposite sides of the circle (Red) near the midpoint of.   Figures 6 (A) -6 (D)-Complete maps of Haemophilus influenzae Rd genome. prediction The coding region is indicated on each chain. The rRNA and tRNA genes are linear and Shown as triangles. Genes are coded by color according to the role category described in the legend Has been Gene identification (GeneID) numbers are the numbers in Tables 1 (a), 1 (b) and 2. Corresponding to Where possible, three letter notation is also provided.   Figure 7-Eight members of the bushy hem gene population present in Haemophilus influenzae type b The region of the chromosome of Haemophilus influenzae containing the gene and the same in the Rd of Haemophilus influenzae Comparison with area. In this region, the pepN and purE genes are flanked by both organisms. ing. However, in the non-infectious Rd strain, the eight genes The child has been cut off. In this region of the Rd strain, a 172 bp spacer region is located. And the pepN and purE genes are flanked.   Figure 8-Hydrophobicity analysis of five predicted channel proteins. Known peptide sequence 5 predicted coding regions that show no homology to (87 GenBank releases) Amino acid sequence, each amino acid sequence is characteristic of a channel-forming protein Are shown. The sequence of the predicted coding region is Uses the WorksWorks software package (Intelligenetics) The Kite-Doolittle algorithm (Kyte and Dooolit) tle, J.M. Mol. Biol.157: 105 (1982)) (with a range of 11 residues).                       Detailed Description of the Preferred Embodiment   The invention is based on the sequencing of the Rd genome of Haemophilus influenzae. One obtained The following nucleotide sequence is provided in SEQ ID NO: 1. As used herein The term “primary front row” refers to a nucleotide sequence represented by IUPAC nomenclature. .   The sequence provided in SEQ ID NO: 1 is found in the Rd genome of Haemophilus influenzae Adapted in relation to unique NotI restriction endonuclease sites. Skilled skills Said that this start / stop point was chosen on delegation and reflects structural significance You will easily recognize that you have not.   The present invention relates to the nucleotide sequence of SEQ ID NO: 1 or a representative fragment thereof. Is provided in a form that can be easily used, analyzed, and interpreted by a skilled technician. One In an embodiment, the sequence corresponds to the nucleotide sequence provided in SEQ ID NO: 1. Is provided as a continuous sequence of primary sequence information.   As used herein, "the nucleotide sequence represented by SEQ ID NO: 1" "Representative fragments" are those that are currently listed in publicly available databases. No sequence identification number: refers to any part of 1. Preferred representative fragment of the present invention Reading frame, expression control fragment, uptake control flag That can be used to diagnose the presence of R. influenzae in the It is a fragment. The identification of such preferred representative fragments is shown in Table 1 (a ) And 2, but are not limited thereto.   The nucleotide sequence information provided in SEQ ID NO: 1 is based on Megabase Shot Sequencing the Rd genome of Haemophilus influenzae using a cancer sequencing method Obtained by. The three parameters for accuracy discussed in the examples below are In use, the inventor has determined that the sequence of SEQ ID NO: 1 has up to 99.98% accuracy. Calculated. Thus, the nucleotide sequence provided in SEQ ID NO: 1 is Nucleotide sequence of Rd genome, not necessarily 100% complete Is very accurate.   As discussed in detail below, SEQ ID NO: 1 and Tables 1 (a) and 2 Using the information provided, along with routine cloning and sequencing methods, Those of ordinary skill in the art will recognize a wide variety of H. influenzae proteins. All important "representative fragments, including quality coding open reading frames (ORFs)" Could be cloned and their sequence determined. Extremely In rare cases, this may be present in the nucleotide sequence disclosed in SEQ ID NO: 1. May reveal errors in the nucleotide sequence. Therefore, once the present invention is used, When it becomes available (that is, the sequence identification number: 1 and the information in Tables 1 (a) and 2 are used once) (When possible), the resolution of the rare error in sequencing in SEQ ID NO: 1 It will be within the skill level of the field. Nucleotide sequence editing software is publicly available Noh. For example, the Applied Biosystem (AB) ) 'S Auto Assembler ™ while visually examining nucleotide sequences Can be used as an auxiliary.   Even if all the very rare sequencing errors of SEQ ID NO: 1 have been corrected The nucleotide obtained is still the nucleotide sequence of SEQ ID NO: 1. Will be at least 99.9% identical.   Genomic nucleotide sequences from different influenza strains may vary slightly. Has become. However, the nucleotide sequence of the genome of all influenza strains The sequence is at least 99.9% identical to the nucleotide sequence provided in SEQ ID NO: 1. There will be.   Thus, the invention further comprises a nucleotide sequence of SEQ ID NO: 1 comprising at least 9 9.9% identical nucleotide sequences are easily used, analyzed and interpreted by skilled technicians Provide in a form that you can. The nucleotide sequence is SEQ ID NO: 1. The method for determining whether a sequence is at least 99.9% identical is routine and It is readily available to skilled technicians. For example, the well-known fasta algorithm (Pearson and Lipman, Proc. Natl. Acad. Soi. USA85: 2444 (1988 ) Can be used to obtain the percent identity of the nucleotide sequence. Computer-related implementation   Nucleotide sequence provided in SEQ ID NO: 1, representative fragment thereof Or nucleotide sequences at least 99.9% identical to SEQ ID NO: 1 It could be "provided" in a variety of media that facilitate use. As used herein Sometimes, provided is the nucleotide sequence of the present invention, that is, SEQ ID NO: 1. Provided nucleotide sequence, representative fragment or SEQ ID NO: 1 Of non-isolated nucleic acid molecules containing a nucleotide sequence at least 99.9% identical to Say things. Such a product may be the Rd genome or a subset thereof. (E.g., Rd reading frame (ORF) for Haemophilus influenzae) Test the Rd genome or a subset of the genome present in natural or purified form A skilled technician tests this product using means not directly applicable to Provide in a form that allows you to do so.   In one application of this embodiment, the nucleotide sequence of the present invention is It can be recorded on a read medium. As used herein, "computer Computer readable media "means that a computer can read and directly access Say medium. Such media include floppy disks and hard disk storage. Storage media and magnetic storage media such as magnetic tapes; optical storage media such as CD-ROMs Body: electronic storage media such as RAM and ROM; and compounds of these categories For example, but not limited to, magnetic / optical storage media. Skilled technician Uses the currently known computer readable media to Computer readable medium with the recorded nucleotide sequence It will be easy to understand how to create a product.   As used herein, "recorded" refers to computer readable media. The process of storing information. Skilled technicians should be able to The method of recording information on the read-out medium is easily adopted to obtain the nucleotide sequence information of the present invention. Would be made.   Creation of a computer-readable medium recording the nucleotide sequence of the present invention Skilled technicians can use a variety of data storage structures to Wear. The choice of data storage structure is generally selected to access stored information. Based on the means chosen. In addition, various data processor programs and Computer-readable nucleotide sequence information of the invention using It can be stored on a delivery medium. Sequence information is stored in a word processing text file. Or Word Perfect (Microsoft Word) or Microsoft Word (Micr o Format in commercially available software such as Soft Word) Or during database applications such as DB2, Sybase, Oracle, etc. It can be shown in the form of a saved ASCII file. The skilled technician must Obtaining a computer-readable medium on which the nucleotide sequence information of Any number of data processor construction formats (eg, text files) File or database) can be easily adapted.   SEQ ID NO: 1 nucleotide sequence, representative fragment or sequence thereof Computer readable nucleotide sequence at least 99.9% identical to 1 Skilled engineers can provide sequence information for various purposes by providing Can be accessed. A skilled technician submits the Computer software that can access the sequence information provided is publicly available. Is available at The following example uses BLAST (Altschul J. et al. Mol. Biol.215: 403-410 (1990)) and BLAZE (Brutlag et al.) , Comp. Chem.17; 203-207 (1993)) Software that executes search algorithms How to use the open reading frame (O) in the Rd genome of Haemophilus influenzae RF), including its homologues or proteins obtained from other organisms. Is shown. Such an ORF is a fragment in the Rd genome of Haemophilus influenzae. Protein that encodes To produce commercially important proteins, such as enzymes used in the manufacture of cereals Useful.   The present invention further relates to systems containing the sequence information described herein, especially Provide a computer-based system. Such a system is Designed to identify commercially important fragments of the Rd genome Is being measured.   As used herein, a "computer-based system" is a computer-based system of the present invention. Hardware means, software used to analyze nucleotide sequence information Air means and data storage means. Computer based system of the present invention The minimum hardware means is a central processing unit (CPU), input means, It includes output means and data storage means. Skilled technicians are currently available Any suitable computer-based system is suitable for use in the present invention It can be easily understood.   As described above, the computer-based system of the present invention Data storage means for storing the sequence of otide and necessary hardware means and It includes software means for supporting and implementing the search means. As used herein The `` data storage means '' can store the nucleotide sequence information of the present invention. Memory or products that record nucleotide sequence information of the present invention. Memory access means that can be used.   As used herein, "search means" refers to a target sequence or target structural motif. Computer to compare the sequence with the sequence information stored in the data storage Refers to one or more programs that run on the based system. Search method Is a fragment of the Rd genome that matches the specific target sequence or target motif. Used to identify fragments or regions. A variety of known algorithms A variety of publicly disclosed and commercially available software for performing search means Air is used and used in the computer-based system of the present invention Can be. An example of such software is MacPattern (E). MBL), BLASTN and BLASTX (NCBIA). Not determined. Skilled technicians have available algorithms to perform homology searches. Any software or executable software package based on the computer of the present invention. It can be easily recognized that it can be adapted for use in the system.   As used herein, a "target sequence" is six or more nucleobases. Tide or any DNA or amino acid of two or more amino acids It can be an acid sequence. Skilled technicians believe that the longer the target sequence, Sequences are less likely to be present as random occurrences in the database. It can be easily recognized. The most preferred sequence length of the target sequence is about 10 to 10 0 amino acids or about 30 to 300 nucleotide residues. However, the Inn Commercially important fragments of the fluenza Rd genome, such as gene expression and Searches for sequence fragments involved in protein processing are shorter length fragments. Is well recognized.   As used herein, “target structural motif” or “target motif” Three-dimensional arrangement in which the sequence (s) is formed by folding the target motif Refers to any reasonably selected sequence or combination of sequences that is selected based on . A variety of target motifs are known in the art. Protein target motif Include, but are not limited to, an enzyme active site and a signal sequence. Nucleic acid Target motifs include promoter sequences, hairpin constructs and inducible expression elements ( Protein binding sequences).   Using a variety of structural formats for input and output Input or output information with the computer-based system of the invention Can be The preferred format for the output means is the target sequence or target. Of the Rd genome of Haemophilus influenzae having various degrees of homology with the genetic motif Rank. What has been shown in this way is available to skilled technicians in varying amounts of target distribution. Provides a ranking of the sequence containing the sequence or target motif, and Identify the degree of homology contained in the fragment.   Compare target sequences or target motifs with data storage using a variety of comparison methods To identify sequence fragments of the Rd genome of Haemophilus influenzae . In embodiments of the present invention, the BLAST and BLAZE algorithms (Altschul et al. J. Mol. Biol.215: 403-410 (1990)) It was used to identify an open reading frame within the Haemophilus influenzae Rd genome. Skilled technicians All publicly available homology search programs are based on the computer of the present invention. It can easily be used as a search method for the system. You can understand.   One application of this embodiment is provided in FIG. FIG. 2 is a diagram for implementing the present invention. Provides an exploded view of a computer system 102 that can be used for Compu The motor system 102 includes a processor 108 coupled to the bus 104. Busbar Has a main memory 108 (preferably implemented as random access memory, RAM And a variety of secondary storage devices 110, such as hard drive 112 and removable A secure media storage device 114 is also coupled. Removable media storage device 114 For example, floppy disk drive, CD-ROM drive, magnetic tape drive It can represent live and the like. Control logic and / or data is recorded A removable storage medium 116 (eg, floppy disk, compact Disk, magnetic tape, etc.) can be inserted into the removable media storage device 114. it can. The computer system 102 may be accessed once by the removable media storage device 114. Control logic and / or from the inserted removable media storage device 114 Includes software suitable for reading data.   The nucleotide sequence of the present invention comprises a main memory 108, an optional secondary storage device 110. And / or may be stored on removable storage medium 116 in well-known fashion. Geno Software that accesses and processes the array of programs (eg, search tools, Comparison tools, etc.) reside in the running main memory. Biochemical embodiment   Another embodiment of the present invention relates to an isolated fragment of the Rd genome of Haemophilus influenzae. Is pointed at. The fragments of the Rd genome of the Haemophilus influenzae of the present invention include , Fragment encoding peptide (hereinafter referred to as open reading frame (ORF)), manipulation A fragment that regulates the expression of the operably linked ORF (hereinafter, expression regulation flag) Fragment (EMF)), a fragment that mediates the uptake of bound DNA fragments into cells. (Hereinafter referred to as uptake regulatory fragment (UMF)) and influenza in the sample. Fragments that can be used to diagnose the presence of Rd. (DF)), but is not limited thereto.   As used herein, "isolated nucleic acid molecule" or "H. influenzae Rd geno What is an "isolated fragment of a system?" Compounds that have been subjected to purification procedures and are usually associated with the composition A nucleic acid molecule having a specific nucleotide sequence in which the number of To tell. A variety of purification means can be used to produce the isolated fragments of the present invention. it can. These separate the components of the solution based on charge, solubility, or size. But not limited thereto.   In one embodiment, the Rd DNA of Haemophilus influenzae is mechanically sheared for 15-20 A fragment of kb length can be made. Then use these fragments Using the above fragment in a lambda clone as described in the Examples below. Generating a Haemophilus influenzae Rd library by inserting Can be. Then use the nucleotide sequence information provided in SEQ ID NO: 1 Thus, for example, a primer with the ORF provided at mark 1 (a) on the side is generated. Can be made. The lambda DNA line was then cloned using the PCR cloning method. The ORF can be isolated from the prairie. PCR cloning technology Well known in the art. Thus, SEQ ID NO: 1, Table 1 (a) and Table 2 are available Given the nature, isolate any ORF or other nucleic acid fragment of the invention Things will be boilerplate.   The isolated nucleic acid molecules of the present invention include single- and double-stranded DNA and single-stranded RNA. But not limited to these.   As used herein, the "open reading frame", ORF, has no stop codon. Refers to a series of three bases that code for different amino acids and can be translated into proteins It is a functional array. Tables 1a, 1b and 2 show ORFs in the Rd genome of Haemophilus influenzae. Has been identified. In particular, Table 1a is based on the organisms in parentheses (see column 4 of Table 1 (a)). Copy the described proteins based on homology that matches the resulting protein sequence. The location of the ORF in the loaded H. influenzae genome is indicated.   The first column of Table 1 (a) provides the "gene identification" for a particular ORF. This information Information is useful for two reasons. First, the inputs provided in FIGS. A complete map of the R. fluenza Rd genome is provided by these gene identification numbers. Has become widespread in ORFs. Second, Table 1 (b) uses the gene identification numbers , Which ORFs have been provided in public databases so far.   Columns 2 and 3 of Table 1 (a) are the nucleotide sequence provided in SEQ ID NO: 1. The position of the ORF inside is shown. For those of ordinary skill, the ORF is You will recognize that they may be pointing in the opposite direction in the Genza genome. This This is shown in columns 2 and 3.   Column 5 of Table 1 (a) shows the protein encoded by the ORF and column 4 Percent identity to the corresponding protein from the organism in parentheses is shown. You.   Column 6 of Table 1 (a) shows the protein encoded by the ORF and column 4 The percentage similarity to the corresponding protein from the organism in parentheses is indicated. You. The concepts of percent identity and percent similarity between two polypeptide sequences are It is well understood in the art. For example, at three amino acid positions (eg, Two polypeptides 10 amino acids in length differing in positions 1, 3, and 5) It is said to have a percent identity of 70%. However, this same two poly Peptides are "similar", for example, in which the amino acid portion at position 5 is not identical, (Ie, have similar biochemical characteristics), a percent similarity of 80% Would be considered to have.   Column 7 of Table 1 (a) shows the length of the amino acid homology match.   Table 2 also shows "homologous matches" with known protein sequences obtained from another organism. Haemophilus influenzae Rd genome encoding unsuccessful polypeptide sequence ORF is provided. Details on the algorithms and criteria used for homology searches Details are further provided in the examples below.   Skilled technicians are responsible for influenza R other than those shown in Tables 1 (a), d ORFs in the genome, for example, using the computer-based system of the present invention Duplicate or conflict with the identified ORF, in addition to those that can be The ORF encoded by the opposite strand can be easily identified.   As used herein, an "expression control fragment", EMF, is an operable fragment. Refers to a series of nucleotide molecules that regulate the expression of an ORF or EMF bound to You.   As used herein, when expression of a sequence is altered by the presence of EMF The sequence is said to "regulate the expression of the operably linked sequence." EMF Motor and promoter regulatory sequences (inducible elements), including but not limited to Not done. One class of EMF responds to specific modulators or physiological events And a fragment that induces the expression of an operably linked ORF. Hemov For a review of known EMFs from the genus Virus, see Tomb et al.104: 1-1 0 (1991)) and Chandler M. S. (Chandler, MS) (Proc. Natl. Aoad. Sci. USA89: 1826-1830 (1992)) You.   The EMF front row depends on the proximity to the ORF provided in Tables 1 (a), 1 (b) and 2. Can be identified in the Rd genome. Table 1 (a), 1 (b) or Is the length of about 10 to 200 nucleotides taken at the 5 'of any one of the two ORFs The intergenic fragment or the fragment of the intergenic fragment In a manner similar to that found in the column, expression of the operably linked 3 'ORF was Will adjust. As used herein, “intergenic fragment” is defined herein as The fragment of the genus Haemophilus between the two ORFs described in the above. Some EMF is a target sequence or target motif in the computer-based system of the present invention. Can be identified using known EMF as the   The presence and activity of EMF can be confirmed using an EMF trap vector. Wear. The EMF trap vector has a cloning site 5 'for the marker sequence. Have. The marker sequence has an identifiable phenotype, such as antibiotic resistance or recruitment The auxotrophic factor is encoded and the EMF trap vector is The above phenotype can be identified or assayed when placed in a suitable host under the circumstances. You. As described above, EMF regulates the expression of an operably linked marker sequence. Will do. More detailed consideration of various marker sequences Is provided below.   A sequence considered to be EMF is replaced with a marker sequence in the EMF trap vector. Cloned in all three open reading frames of one or more restriction sites upstream from I do. This vector is then transformed into a suitable host using known methods. And the phenotype of the transformed host is tested under appropriate conditions. As mentioned above , EMF regulates the expression of an operably linked marker sequence.   As used herein, an "uptake regulatory fragment", UMF, binds DN A series of nucleotide molecules that mediate the uptake of the A fragment into cells You. UMF uses the computer-based system described above to target sequence or target. Can be easily identified using a known UMF as the genetic motif.   The presence and activity of UMF allows what is considered UMF to bind to the marker sequence. Can be confirmed. Next, the obtained nucleic acid molecule is transferred to a suitable host under appropriate conditions. And measure the uptake of the marker sequence. Above Thus, UMF increases the frequency of uptake of the bound marker sequence. Haemophilus For a review of DNA uptake in Goodgal S. H. (Goodgall, SH) J. et al. Bact.172: 5924-5928 (1990).   As used herein, "diagnostic fragment", DF, A sequence of nucleotide sequences that selectively hybridizes to a sequence. DF Is by identifying unique sequences within the Haemophilus influenzae Rd genome or , In a suitable diagnostic format to measure amplification or hybridization selectivity Generating and testing a probe or amplification primer consisting of the F sequence Can be easily identified.   Sequences that fall within the scope of the present invention are not limited to the particular sequences described herein. It also includes those alleles and species variants. Alleles and variants are sequence knowledge SEQ ID NO: 1, a representative fragment thereof or a sequence identifier number: 1 At least 99.9% identical nucleotide sequence to another isolate of the same species Can be routinely determined by comparing with the sequence obtained from Further In order to accommodate codon variability, the present invention provides certain O 2 compounds disclosed herein. Nucleic acid molecules that encode the same amino acid sequence as that encoded by RF are included. In other words, one codon replaces the same amino acid in the coding region of the ORF. It is expressly intended to replace it with another coding codon.   Any particular sequence disclosed herein may be a specific fragment, such as an ORF. Re-sequencing the sequence in both directions (ie, the front row of both strands) Can be screened. Alternatively, screening for errors is a Isolated using some or all of the fragments in question as primers or primers By determining the sequence of the corresponding polynucleotide of Haemophilus influenzae origin, Can be implemented.   Each ORF of the Haemophilus influenzae Rd genome disclosed in Tables 1 (a), 1 (b) and 2 And EMF found in the 6 'of the ORF can be used as a polynucleotide reagent in a number of ways. Can be used with These sequences are very similar to the H. influenzae RD in the sample. Diagnostic probe or diagnostic amplification primer to detect the presence of specific microorganisms such as Can be used. This is very selective for Haemophilus influenzae. This is particularly true in the case of fragments or ORFs.   In addition, triple helix formation using the broadly described fragments of the invention Alternatively, gene expression can be controlled by antisense DNA or RNA. And both of these methods involve binding of a polynucleotide sequence to DNA or RNA. Is based on Polynucleotides suitable for use in these methods are typically 20 Gene region that is 40 bases in length and involved in transcription (triple helix-Lee, etc.) , Nucl. Acids Res.6: 3073 (1979); Cooney et al., Science241: 456 (1988 ); And Dervan et al., Science.2511360 (1991)) or the mRNA itself (A Antisense-Okano, J.M. Neurochem.56: 560 (1991); Oligodeoxynucleot ides as Antisense Inhibitors of Gene Expression, CRC Press, Flow Designed to be complementary to Boca Leton, Lida (1988). Triple helix Formation maximizes transcription of RNA from DNA. Stops appropriately while antisense RNA hybridization forms Blocks translation into peptides. Both technologies proved to be effective in the model system Have been. The information contained in the sequences of the invention may be antisense or triple helix Necessary for designing lignonucleotides.   The invention further relates to one or more of the Haemophilus influenzae Rd genomes of the invention. A recombinant construct comprising the fragment is provided. The recombinant construct of the present invention is a vector For example, plasmids or viral vectors, and contains Fluenza Rd is inserted in the forward or reverse direction. One of the ORFs of the present invention In the case of a vector containing one, the vector may be further manipulated, for example, into an ORF. Regulatory sequences can be included, including operably linked promoters. Book In a vector comprising EMF and UMF of the invention, the vector may further comprise EMF or UMF. May comprise a marker sequence or a heterologous ORF operably linked to . Many suitable vectors and promoters are known to those of skill in the art. And can be obtained commercially to produce the recombinant constructs of the present invention. Wear. The following vectors are provided as examples. Bacterial: pBs, phage script, PsiX174, pBluescript SK, pBS KS, pNH8a, pNH16a, pNH18a, p NH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRI T6 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTI, pS G (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).   The promoter region is based on OAT (chloramphenicol transferase). Using a vector or other vector with a selectable marker to select from the desired gene. You can choose. Two suitable vectors are pKK232-8 and pCM7. Special Other named bacterial promoters include lacI, lacZ, T3, T7, gpt, Lambda P_EAnd trc. Eukaryotic promoters include CMV First, HS V thymidine kinase, early and late SV40, LTR obtained from retrovirus As well as mouse metallothionein-I. Appropriate vector or promoter The choice is just within the normal level of skill in the art.   The present invention further relates to an isolated fragment of the Rd genome of the Haemophilus influenzae of the present invention. Providing a host cell containing at least one of the fragments, wherein the fragment Have been introduced into host cells using the method. The host cell is like a mammalian cell A higher eukaryotic host cell, such as a yeast cell. Alternatively, the host cell can be a prokaryotic cell, such as a bacterial cell. Host Introduction of the recombinant construct into the cells was performed by calcium phosphate transfection, DE By AE, dextran mediated transfection or transduction (Davis, L., et al., Basic Methods in Molecular). Biology (1986)).   A host cell containing one of the fragments of the Rd genome of the Haemophilus influenzae of the present invention. Gene products encoded by isolated fragments using cells in a conventional manner (For ORFs) or under the control of EMF It can be used to produce proteins.   The present invention further relates to a nucleic acid fragment of the present invention or a nucleic acid fragment of the present invention. Isolated polypeptides encoded by degenerate variants of the polypeptide. "Shrink By "heavy variant", a nucleic acid fragment (eg, an ORF) of the present invention can be Peptide sequences differ, but the same polypeptide sequence is copied due to the degeneracy of the genetic code. A coding nucleotide fragment is contemplated. Preferred nucleic acids of the present invention The fragment is the ORF shown in Table 1 (a) that encodes the protein.   The isolated polypeptides of the invention can be prepared using a variety of methodologies known in the art. Either one of protein or protein can be obtained. At the easiest level Can synthesize amino acid sequences using commercially available peptide synthesizers. Wear. This is the production of smaller peptides and fragments of larger polypeptides. Is particularly useful in Fragments include, for example, antibodies to the native polypeptide Is useful in the production of In an alternative, the polypeptide or protein is a polypep Purified from bacterial cells that naturally produce tides or proteins. In the technical field The skilled artisan can readily follow known methods for isolating polypeptides and proteins Of an isolated polypeptide or protein You can get one. These include immunochromatography, HPLC , Size exclusion chromatography, ion exchange chromatography and immunoa Including, but not limited to, affinity chromatography.   The polypeptide or protein of the present invention may alternatively be a desired polypeptide or protein. It can be purified from cells that have been modified to ring the protein. This specification As used herein, cells are not normally produced or cells are usually at lower levels Genetic manipulation so that cells produce the polypeptide or protein produced in When the cells have been modified to express the desired polypeptide or protein, Say that it has been modified. Those skilled in the art will appreciate the polypeptides or proteins of the present invention. Recombination sequences in eukaryotic or prokaryotic cells to produce cells that produce one of the proteins The method of introduction and expression of either or synthetic sequences can be readily adapted. Can be.   Using any host / vector system, one or more ORFs of the invention Can be expressed. These include eukaryotic hosts such as HeLa cells, Cv- 1 cell, COS and Sfs cells, and prokaryotic hosts such as E. coli and Bacillus Bacteria include, but are not limited to. Most preferred cells are usually Does not express a polypeptide or protein or a polypeptide or protein Cells that express proteins at low natural levels.   As used herein, “recombinant” refers to a group of polypeptides or proteins. Derived from a recombinant (eg, microbial or mammalian) expression system . A “microorganism” is a recombinant microorganism produced in a bacterial or fungal (eg, yeast) expression system. Refers to lipetide or protein. As a product, "recombinant microorganisms" Possibly essentially free of endogenous substances and without associated natural glycosylation Specify the peptide or protein. In most bacterial cultures, for example in E. coli The expressed polypeptide or protein has no glycosylation modifications; The polypeptide or protein expressed in the mother is expressed in mammalian cells Has a different glycosylation pattern.   "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides . Generally, DNs encoding the polypeptides and proteins provided herein. A fragment is a set containing regulatory elements derived from a microbial or viral operon. In order to provide a synthetic gene that can be expressed in the recombinant transcription unit, From a fragment of the Rd genome and a short oligonucleotide linker, or Assembled from a series of oligonucleotides.   "Recombinant expression vehicle or vector" refers to a polypeptide (polypeptide) from a DNA (RNA) sequence. Plasmid or phage or virus or vector for expressing . The expression vehicle may comprise (1) one or more genes having a regulatory role in gene expression. Child elements, such as promoters or enhancers, (2) transcribed into mRNA and Structure or coding sequence that is translated into a protein, and (3) appropriate transcription initiation And a transcription unit comprising an assembly of termination sequences. Structural units intended for use in yeast or eukaryotic expression systems are preferably Contains a leader sequence that enables extracellular secretion of translated protein in the host cell. Have. Alternatively, the recombinant protein is expressed without a leader or transport sequence If so, it can contain an N-terminal methionine residue. This residue is the last Subsequent cleavage from the expressed recombinant protein to provide the product Can or cannot.   "Recombinant expression system" integrates a recombinant transcription unit stably into chromosomal DNA. Refers to a host cell that is running or has a recombinant transcription unit extrachromosomally . The cells can be prokaryotic or eukaryotic cells. Recombinant expression identified herein The system is based on the induction of regulatory elements associated with the expressed DNA fragment or synthetic gene. To express a heterologous polypeptide or protein.   Mature proteins can be obtained from mammalian cells, yeast, bacteria, Alternatively, it can be expressed in other cells. Using a cell-free translation system, Producing such proteins using RNA derived from NA constructs Can also be. Cloning and suitable for use in prokaryotic and eukaryotic hosts Expression vectors include molecular cloning, such as Sambrook, etc .: Laboratory Manual (Molecular Cloning: A Laborato ry Manual, 2nd edition, Cold Spring Harbor, New York (198), the disclosure of which is incorporated herein by reference. It is described.   In general, recombinant expression vectors contain an origin of replication and a source that allows transformation of the host cell. And selectable markers such as the E. coli ambicillin resistance gene and S-cerevisiae T Directs transcription of downstream structural sequences derived from the RP1 gene and highly expressed genes Contains a promoter. Such promoters are, inter alia, 3- Sphoglycerate kinase (PGK), factor a, acid phosphatase or heat shock Can be derived from operons that encode glycolytic enzymes such as glycoproteins. Wear. Heterologous structural sequences include translation initiation and termination sequences, (and preferably Or with a leader sequence capable of directing secretion of the translated protein into the extracellular medium Assembled in different phases. Optionally, the heterologous sequence has a desired characteristic, such as an expressed set. Fusions containing N-terminal identifying peptides that stabilize the recombinant product or simplify purification Can encode a protein.   Expression vectors useful for use in bacteria are operable having a functional promoter. Within the active reading phase, together with the appropriate translation initiation and termination signals, It is constructed by inserting a structural DNA sequence that encodes quality. Vector Provide amplification in the host to ensure vector maintenance and, if desired, To include one or more phenotypic selectable markers and origins of replication. I have. Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, and Salmonella typhimurium. And various species within the genus Pseudomonas, Streptomyces and Staphylococcus , But others can be used as selections.   In an exemplary embodiment, an expression vector useful for use in bacteria is a selectable marker. And the genetic elements of the well-known cloning vector pBR322 (ATCC 37017) Contains bacterial origin of replication derived from commercially available plasmids containing However, the present invention is not limited to this example. Such commercially available vectors include, for example, , PKK223-3 (Pharmacia Fine Chemicals, Ufsala, Sweden) and GEM1 (Promega Biotec, Ma, Wisconsin, USA Dison). These pBR322 "backbone" sections are Combined with the motor and the structural sequence to be expressed.   After transforming the appropriate host strain and growing the host strain to an appropriate cell density, The selected promoter can be replaced by appropriate means (eg, temperature shift or chemical induction). Thus, it is activated and the cells are cultured for a further period. Cells are typically centrifuged Collected, destroyed by physical or chemical means, and the resulting crude extract Is maintained for further purification.   Various mammalian cell culture systems can also be used to express recombinant proteins. You. Examples of mammalian expression systems include Gluzman, Cell.twenty three: 175 (1 981) monkey kidney fibroblast cell line COA-7 and compatible Other cell lines capable of expressing the vector, such as C127, 3T3, CHO, HeLa and BHK cell lines are included. Mammalian expression vectors have an origin of replication, a suitable promoter And enhancers, and even necessary ribosome binding sites, polyadenylation Sites, splice donor and acceptor sites, transcription termination sequences, and 5 ' May contain transcribed sequences. DNA sequence derived from SV40 virus genome, eg For example, SV40 origin, early promoter, enhancer, splice and polyadenylate The site of cleavage can be used to provide the required non-transcribed genetic elements.   Recombinant polypeptides and proteins produced in bacterial cultures are usually Can be used for more salting out, aqueous ion exchange or size exclusion chromatography steps. Prior to initial extraction from the cell pellet, it is isolated. Completed mature protein placement In doing so, a protein regeneration step can be used if necessary. last Use high performance liquid chromatography (HPLC) for the final purification step. Can be. The microbial cells used for protein expression were subjected to a freeze-thaw cycle. By any convenient method, including the use of sonication, mechanical disruption or cell lysis. Can be destroyed.   The present invention further provides an isolated polypeptide substantially equivalent to that described herein, Includes proteins and nucleic acid molecules. As used herein, substantially equivalent means Different from the reference sequence by one or more substitutions, deletions or additions And the net effect is that unfavorable functional differences between the reference and target sequences Both non-contributing nucleic acid and amino acid sequences, e.g. Can be. For the purposes of the present invention, having equivalent biological activity and equivalent expression characteristics Are considered substantially equivalent. For the purpose of equivalence determination, a portion of the mature sequence Disconnection should be ignored.   The present invention further relates to the fragment of the Rd genome of the Haemophilus influenzae of the present invention. Homologs from other strains of S. fluenzae and encoded by the ORFs of the invention Provided is a method for obtaining a homolog of a known protein. As used herein The sequence or protein of Haemophilus influenzae is the Rd gene of Haemophilus influenzae of the present invention. Encoded by one of the nome fragments or one of the ORFs of the invention If it shares significant homology with protein A, this is the influenza of the invention. A fragment encoded by one of the fragments or ORFs of the bacterial Rd genome Identified as homologues of proteins. Specifically, the sequences disclosed herein can be probed Or used as primers and PCR cloning method Using techniques such as dark hybridization, those skilled in the art You can get the body.   As used herein, two nucleic acid molecules or proteins are those two % (Amino acid or nucleic acid) "Share significant homology."   Nucleotide sequence provided by SEQ ID NO: 1 or as few as SEQ ID NO: 1 Region-specific primers or primers derived from nucleotide sequences that are 99.9% identical to each other. The lobes are used to prime DNA synthesis and RNA amplification, and (Innis et al., PCR Protocols, Academic Press, Sande, CA Includes cloned DNA encoding homologs using Diego (1990) Colonies to be identified can be identified.   SEQ ID NO: 1 or a nucleotide at least 99.9% identical to SEQ ID NO: 1 When using primers derived from the sequence, those skilled in the art By using stringent conditions (eg, annealing at 50-60 ° C.) Recognizes that only sequences that are 75% or more homologous to the Will be. Use lower stringency conditions (eg, annealing at 35-37 ° C). The use of this primer will also amplify sequences that are more than 40-50% homologous to this primer. There will be.   SEQ ID NO: 1 or sequence identification for colony / plaque hybridization DNA sequence derived from a nucleotide sequence at least 99.9% identical to No. 1 When using a probe, those skilled in the art will recognize that high stringency conditions (eg, 5 × S Hybridization at 50-65 ° C. in SPC and 50% formamide and 0.5 × SS By washing with 50-65 ° C in PC), more than 90% Sequences having the same region can be obtained, and lower stringency conditions (eg, Hybridization with 5X SSPC and 40-45% formamide and SSP More than 35-45% homology to this probe by using It will be appreciated that sequences with different regions will be obtained.   An organism naturally expresses such a protein or expresses such a protein. Any source of homologues of the present invention as long as it contains the encoding gene Organisms can be used. The most preferred organism for isolating a homolog is It is a bacterium closely related to Haemophilus influenzae Rd. Use of the composition of the present invention   Each ORF provided in Table 1 (a) is referred to as Relay. (Riley, M.) (Microbiol ogy Reviews57(4): 862 (1993)) 102 biological role categories Assigned to one of the This allows the skilled technician to identify each coding sequence identified. The use can be determined. Table 1 (a) is further coded by each ORF Provides confirmation of the type of peptide. As a result, those skilled in the art The polypeptides of the invention can be purchased commercially, consistent with the type of putative polypeptide identification, Can be used for therapeutic and industrial purposes.   Such identification allows those skilled in the art to identify the H. influenzae ORF. , In a manner similar to the known type of sequence being identified; Used to ferment sources or produce specific metabolites can do. (For a review of enzymes used within for-profit industries, see Biohem ical Engineering and Biotechnology Handbook 2nd edition, Macmillan Publ . Ltd., New York (1991) and Biocatalysts in Organic Syntheses Edit J. Tramper et al., Elsevier Science Publishers, Amste, The Netherlands Ledame (1985)).   1. Biosynthetic enzymes   Enzymes involved in the breakdown of metabolic intermediates, enzymes involved in central intermediate metabolism, Enzymes involved in respiration, both aerobic and anaerobic enzymes involved in fermentation, ATP proto Enzymes involved in transduction of amino acids, enzymes involved in a wide range of regulatory functions, and amino acid synthesis Enzymes involved in DNA synthesis, enzymes involved in nucleotide synthesis, cofactors and vitamin synthesis Catalytic reactions involved in intermediate and macromolecule metabolism, including the production of small molecules Reads encoding proteins involved in mediation of synthesis, cellular processes and other functions The framework can be used for industrial biosynthesis. Present in Haemophilus The various metabolic pathways are based on the absolute nutritional requirements as well as the various identified in Table 1 (a). Can be identified by testing the enzyme.   The ORF identified in the intermediate metabolism category and identified in Table 1 (a) A large number of proteins are loaded for degradation of intermediate metabolites and non-macromolecular metabolism. Are involved in Some of the enzymes identified include amylase, glucose, and oxy. Including dase and catalase.   Proteolytic enzymes are another class of commercially important enzymes. Tampa Enzymes are used in a number of industrial processes, including the processing of flax and other plant fibers, in juice. Extraction, clarification and pectin removal, extraction of vegetable oils, and Use in softening fruit and plants has been found. Tampa used in the food industry A detailed review of pyrolytic enzymes can be found in Rombouts et al. (Symbiosistwenty one: 79 (1 986)) and Voragen et al. (Biocatalyst in Agricultural Biot) echnology, edited by JR. Whiteter et al., American Chemical Society Sympos ium Seriess389: 93 (1989)) Have been. The metabolism of glucose, galactose, fructose and xylose is of the genus Haemophilus Is an important part of the early metabolism. The enzymes involved in the breakdown of these sugars are industrial Can be used in fermentation. Some of the sugar-converting enzymes that are important from a commercial perspective include Sugar isomerases such as glucose isomerase are included. Ketogulonic acid (K Commercial use for other metabolic enzymes such as glucose oxidase that produce GA) Have been found. KGA is based on the method of Reichstein (Kr ueger, Biotechnology6(A), Rhine, edited by HJ, Verlag Press, de For commercial production of ascorbic acid using Weinheim, Germany (1984) Intermediate.   Glucose oxidase (GOD) is commercially available and in beer It is used in purified as well as fixed form for oxygen removal. Hartmeyer tmeir), etc. (Biotechnology Letters1: 21 (1979). The most important GO The application of D is an industrial scale fermentation of gluconic acid. The market for using gluconic acid is Detergent, textile, leather, photography, pharmaceutical, food, feed and concrete industries (B Edited by igelis, Gene Manipulations and Fungi, Benett JW, etc., Academic  Press, New York (1985), p. 357). GOD in addition to industrial applications Are drugs for the quantitative determination of glucose in body fluids, more recently starch and cellulose Application found in biotechnology for analyzing syrups obtained from digests Have been. Owusu et al. (Biochem. At Biophysics. Acta.872: 83 (1986)).   The main sweetener used today in the world comes from sugar beet and sugar cane It is sucrose. In the field of industrial enzymes, the glucose isomerase method is the market today Shows the largest spread. First, soluble enzymes are used, and then Immobilized enzymes have been developed (Krueger et al., Biotechnology, The Textbook of Ind ustrial Microbiology, Sinauer Associated Incorporated, Mass. Sunderland, Switzerland (1890)). Today, from glucose using immobilized enzymes The use of manufactured high fructose syrup is by far the largest industrial business. this A review of the industrial use of enzymes by Jorgensen (Jorg ensen) (Starch40: 307 (1988)).   Proteinases, such as alkaline serine proteinases, are used as detergent additives. As used, it represents one of the largest amounts of microbial enzymes used in the industrial field. doing. Use of these enzymes in industrial processes because they are of industrial importance There is a lot of published and unpublished information about. (Faultman et al., Acid Prot eassa Structure Function and Biology, edited by Tang, J., Plenum Press New York (1977) and Godfrey et al., Industrial Enzymes, MacMilla. n Publishers, Surrey, UK (1983) and Hepner et al., Report Industrial E nzymes by 1990, Hel Hepner & Associates, London (1986)).   Another class of commercially available proteins of the invention is identified in Table 1. Microbial lipase (Macrae et al., Philosophical Transactions of the C hirai Society of London310: 227 (1985) and Poserke, Journal of the  American Oil Chemist Society611758 (1984)). Lipase Neutralization using lipase-catalyzed interesterification of readily available triglycerides It is mainly used in the fats and oils industry to produce lyserides. To apply lipase, Includes use as a detergent additive to facilitate removal of fat from fabric during the cleaning process You.   Enzymes, and especially microbial enzymes, as key steps in the synthesis of complex organic molecules Use is gaining in popularity at a very fast rate. One very important area is key Production of ral intermediate. The production of chiral intermediates is a wide range of synthetic chemists, especially new drugs Important for scientists involved in the production of pesticides, fragrances and flavors. ( Davies et al., Recent Advances in the Generation of Chirality Entermediat es Using Enzymes, CRC Press, Pocalemton, Florida (1990) . The following reactions catalyzed by enzymes are important for organic chemists: Carbo Hydrolysis and esterification of acid esters, phosphate esters, amides and nitriles Reaction, trans-esterification reaction, amide synthesis, alkanone and oxoalkane Reduction, oxidation of alcohols to carbonyl compounds, sulfides to sulfo Oxidation to oxide, as well as aldol A carbon bond forming reaction such as a reaction. For biotransformation and organic synthesis When considering the use of an enzyme encoded by one of the ORFs of the invention Consider the advantages and disadvantages of using microorganisms, completely different from isolated enzymes Is sometimes necessary. On the one hand whole cell lines or on the other hand partially purified The pros and cons of using an isolated enzyme are described in Bud et al. (Chemistry in B ritain (1987), p. 127).   Aminotransferase, an enzyme involved in the biosynthesis and metabolism of amino acids, is Useful in the catalytic production of carboxylic acids. The advantage of using a microbial-based enzyme system is amino Transferase enzymes catalyze the stereoselective synthesis of l-amino acids only and generally A high catalyst speed. Amino for amino acid production Instructions for using transferase are provided by Roselle-David (Methods of Enzymology136: 479 (1987)).   Another Useful Protein Encoded by the ORF of the Invention The various categories include enzymes involved in nucleic acid synthesis, repair and recombination. Commercial Diverse Enzymes Have Been Isolated from Members of Haemophilus Species . These include Hinc II, Hind III and Hinf I restriction endonucleases. It is. Table 1 (a) shows that the restriction enzymes have direct uses in the biotechnology industry. A wide array of enzymes such as elemental, ligase, gyrase and methylase are identified.   2. Antibody production   As described herein, the proteins of the invention, as well as their homologs, A variety of proteins currently applied to other proteins and known in the art Can be used in procedures and methods. The protein of the present invention further comprises It can be used to generate antibodies that bind selectively. Such anti Antibodies can be either monoclonal or polyclonal, as well as these antibodies. It can be in the form of a fragment and a humanized form of the body You.   The present invention further provides antibodies that selectively bind to one of the proteins of the present invention, A hybridoma producing the antibody is provided. Hybridomas are specific It is an immortalized cell line that can secrete ronal antibodies.   Generally, it produces polyclonal and monoclonal antibodies and the desired antibodies. Techniques for making the resulting hybridomas are well known in the art (Campbell, A .M., Monononal Antibody Technology: Laboratory Technologies in Bio chemistry and Molecular Biology, Elsevier Science Publishers, Ora Amsterdam, the Netherlands (1984); St. Groth et al., J. Immunol. Methods35: 1-21 (1980); Kohler and Milstein, Nature256: 495-497 (1975)) Lioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Toda) yFour: 72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).   Animals known to produce antibodies (mouse, rabbit, etc.) It can be immunized with a peptide. Immunization techniques are well known in the art. this Such methods include subcutaneous or intraperitoneal injection of the polypeptide. Technical field One skilled in the art will recognize that the tandems encoded by the ORFs of the present invention used in immunization methods. Amount of protein varies based on animal to be immunized, antigenicity of peptide and site of injection You will be aware that   Proteins used as immunogens are modified to increase the antigenicity of the protein. It can be corrected or administered in an adjuvant. Increase protein antigenicity Methods for determining antigens are well known in the art and include antigens and heterologous proteins. (Eg, globulin or β-galactosidase) or Including, but not limited to.   For monoclonal antibodies, remove spleen cells from immunized animals and Fused with an eroma cell, such as SP2 / 0-Ag14 myeloma cell, and It can be a hybridoma cell producing a lonal antibody.   The desired features can be obtained using any one of a number of methods well known in the art. A hybridoma cell producing an antibody having the following can be identified. These include ELISA, Western blot analysis or radioimmunoassay. Screening of hybridomas (Lutz et al., Exp. Cell Re. s.175: 109-124 (1988).   Hybridomas secreting the desired antibody are cloned and known in the art. Class and subclass using the methods described (Campbell, A.M. , Monoclonal Antibody Technology: Laboratory Techniques in Biochem istry and Molecular Biology, Elsevier Science Publishers, Netherlands Country Amsterdam (1984)).   The method described for producing single chain antibodies (U.S. Pat. It can be adapted to produce single chain antibodies to the light proteins.   For polyclonal antibodies, from animals immunized with antisera containing the antibody The presence of an antibody having the desired specificity, isolated and using one of the methods described above Screen for   The invention further provides the above-described antibodies in a labeled form for detection. Antibodies include radioisotopes, affinity labels (e.g., biotin, avidin, etc.), enzymes Labels (e.g., horseradish peroxidase, alkaline phosphatase Etc.), fluorescent labels (e.g., FITC or rhodamine, etc.), paramagnetic atoms, etc. Can be labeled for detection. The way to achieve such labeling is It is well known in the art and is described, for example, in (Sternberger, LA, et al., J. Histochem. . Cytochem.18: 315 (1970); Bayer, EA, et al., Meth. Enzym.62: 308 (197 Engval, E. et al., Immunol.109: 129 (1972); Goding, JW, J.M. I mmunol. Meth.13: 215 (1976)).   The labeled antibody of the present invention expresses a fragment of the Rd genome of Haemophilus influenzae. In vitro, in vivo, and in situ assays to identify living cells or tissues Can be used with Say.   The present invention further provides the above antibody immobilized on a solid support. Such a solid support Examples of carriers include plastics such as polycarbonate, agarose and cellulose. Complex carbohydrates such as fallose, acrylics such as polyacrylamide As well as latex beads. Techniques for binding antibodies to such solid supports Techniques are well known in the art (Weir, DM, et al., "Handbook of Experiment"). al Immunology, 4th Edition, Blackwell Scientific Publications, Ock, UK Sford, Chapter 10 (1986); Jacoby, WD, et al., Meth. Enzym.34 Academic  Press, New York (1974)). The immobilized antibody of the present invention is a protein of the present invention. , In vivo and in situ assays and immunoaffinity purification of Can be used in law.   3. Diagnostic assays and kits   The present invention further provides for the use of one of the DFs or antibodies of the present invention in a test sample. Methods are provided for identifying the expression of one of the ORFs of the invention or homologs thereof.   In particular, such a method involves the testing of one or more antibodies or one or more antibodies. Incubate with one or more DFs of the invention, and DF or Assaying the binding of the antibody to a component in the test sample.   Conditions for incubating DF or antibody with a test sample vary. Incu Conditions are the format used in the assay, the detection method used and the assay Depends on the type and nature of the DF or antibody used in the method. Those skilled in the art Any of the commonly available hybridization, amplification or immunoassay formats Can be readily adapted for use with the DFs or antibodies of the present invention. Will be. An example of such an assay is Chardity. (Chard, T.) (An  Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986)); Baroque G . R. (Bullock, GR), et al. (Techniques in Immunocytochemistry, Ac ademic Press, Orlando, Florida, Volume 1 (1982), Volume 2 (1983), Volume 3 (1 985)); (Tijssen, P.) (Practice and Theory of Enzyme Immunoassays: Laboratory Technologies in Biochemistry and Mole cular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985)) Can be.   The test sample of the present invention includes cells, cell proteins or membrane extracts, or biological Target fluids, such as sputum, blood, serum, plasma or urine. Use with the above mentioned oil The test sample used depends on the assay format, the nature of the detection method, and the sample to be assayed. Will vary based on the tissue, cells or extracts used. Cellular protein extraction Methods for preparing exudates or membrane extracts are well known in the art, and the systems used Can be easily adapted to obtain samples compatible with   In another embodiment of the present invention, the assays required to perform the assays of the present invention. Kits containing the drug are provided.   In particular, the invention relates to (a) a first volume comprising one of the DFs or antibodies of the invention. And (b) one or more of the following reagents: washing reagent, bound DF or anti- One or more other containers containing reagents capable of detecting the presence of the body Compartment kit for receiving and enclosing one or more containers in close proximity I will provide a.   In particular, compartment kits include any kit in which reagents are in separate containers. included. Such containers include small glass containers, plastic containers or Tick pieces or paper pieces are included. Such containers allow the sample and reagent to Transporting trials efficiently from one compartment to another so as not to be contaminated And dispensing reagents or solutions from each container from one compartment to another Can be added in a specific manner. Such containers have the capacity to receive test samples. Containers, containers containing antibodies used in the assay, washing reagents (eg, phosphate buffered saline) Container containing saline solution, Tris buffer, etc.) and detect bound antibody or DF A container containing the reagents used for   The type of detection reagent includes a labeled nucleic acid probe, a labeled second antibody, Alternatively, if the first antibody is labeled, it may react with an enzyme or labeled antibody. Antibody binding reagents. Those skilled in the art will appreciate the DF disclosed in the present invention. And one of the established kit formats in which the antibody is well known in the art It will be readily recognized that it can be easily incorporated into   4. Screening assay for binding substances   Using the isolated protein of the present invention, the present invention further provides one of the ORFs of the present invention. Thus, the encoded protein or the Haemophilus geno described herein. To obtain and identify substances that bind to one of the fragments .   Specifically, the above method includes the following steps:     (A) the substance is encoded by one of the ORFs of the invention             Isolated proteins or isolated fragments of the genome of Haemophilus             Contact with the client; and     (B) the substance binds to the protein or the fragment;             Measure whether they match.   Substances screened in the above assays include peptides, carbohydrates, vitamins It can be, but is not limited to, a derivative or other medicament. these Substances can be randomly selected and screened or protein It can be reasonably selected or designed using modeling techniques.   In a random screen, substances such as peptides, carbohydrates, Tamper selected randomly and they are encoded by the ORF of the present invention Assayed for their ability to bind to proteins.   Alternatively, the materials can be rationally selected or designed. As used herein Sometimes, when a substance is selected based on a particular protein configuration, Choose or design rationally. " For example, those skilled in the art A possible method is to generate peptides, pharmaceuticals, etc. that can bind to specific peptide sequences. And rationally designed anti-peptide peptides (eg, Hurby et al., Application of Synthetio Peptides: Antisense Peptides, I n Synthetic Peptides, A User's Guide, WH Freeman, New York ( 1992), 288-307, and Kaspczak et al., Bio. chemistry28: 9230-9238 (1989)) or can produce pharmaceuticals etc. Wear.   In addition to the foregoing, the present invention has been described using one of the broadly described classes of materials of the present invention. Gene expression can be controlled in conjunction with one of the light ORFs or EMF. As noted above, such substances may be screened at random or Can be designed / selected. By targeting the ORF or EMF, The skilled artisan will either rely on a single ORF that relies on the same EMF for expression control, or Design sequence-specific or element-specific substances that regulate the expression of either of multiple ORFs can do.   One class of DNA binding substances is by binding to DNA or RNA. Contains base residues that form a hybrid or form a triple helix Substance. Such substances are the classic phosphodiesters, Diverse sulfides that can be based on thiophene or have base-binding ability It can be ril or a polymer derivative.   Materials suitable for use in these methods typically have 20 to 40 bases, and And a gene region related to transcription (triple helix-Lee et al., Nucl. Acids Res.6: 3073 (1979); Cooney et al., Science241: 456 (1988); and Dervan et al., Scien ce251: 1360 (1991)) or the mRNA itself (antisense-Okano, J. . Neurochem.56: 560 (1991); Oligodeoxynucleotides as Antisense Inhi bitors of Gene Expression, CRC Press, Boca Raton, Florida (1988 Year)). Triple helix formation is the conversion of DNA to RNA Terminates transcription while antisense RNA hybridization forms a Blocks translation to the repeptide. Both technologies prove effective in model systems Has been stated. The information contained in the sequences of the invention may be antisense or triple helix. Necessary for designing oligonucleotides and other DNA binding agents.   A substance that binds to a protein encoded by one of the ORFs of the present invention is By regulating the activity of the protein encoded by the ORF It can be used as a diagnostic agent when controlling bacterial infection. Book Known substances that bind to proteins encoded by one of the ORFs of the invention Physicians who use the technology to control the growth and infection of Haemophilus A medicinal composition can be manufactured.   5. Vaccines and pharmaceutical compositions   The invention further provides for the growth of Haemophilus influenzae or another related organism in vivo or in vivo. Provide medicinal products that can be used to adjust in vitro. As used herein The "pharmaceutical" can be formulated using known techniques to provide a pharmaceutical composition. Defined as a composition. As used herein, "a pharmaceutical of the present invention" means Derived from the protein encoded by the ORY of the invention or Refers to a medicament that is a substance identified using the assays described herein.   As used herein, a medicament may be a growth rate, division rate or survival of the organism in question. When reducing the toxicity, the medicinal product "inhibits the growth of Haemophilus sp. Or related organisms. Adjusted in vivo or in vitro ". Implement the use of the medicament of the present invention It is not necessary to understand the mechanism underlying the action, The article can regulate the growth of an organism in a number of ways. Important for some pharmaceuticals By binding to the protein, thereby blocking the biological activity of the protein Regulate growth while other drugs bind to components on the outer surface of the organism. To block adhesions or make organisms more accessible to multiple natural immune systems Sometimes. Alternatively, the medicament is encoded by one of the ORFs of the present invention Contains proteins and can serve as a vaccine. Outer membrane components, such as For example, the development and use of LPS-based vaccines is well known in the art.   As used herein, a "related organism" is defined by one of the pharmaceutical agents of the present invention. Refers to any organism that can regulate growth. Generally, such organisms are called vaccines Contains homologues of proteins that are targets for drugs or proteins used as You. As such, the relevant organism need not be a bacterium, but a fungus or virus. Pathogens.   The medicaments and pharmaceutical compositions of the present invention may be administered in conventional manner, e.g., orally, topically, intravenously. It can be administered by the intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route. Medicine The composition is administered in an amount effective to treat and / or prevent a particular indication. one Generally, the pharmaceutical composition will be administered in an amount of at least about 10 μg / kg of body weight and In each case, it is administered in an amount not to exceed about 8 mg / kg of body weight per day. In most cases, The dose should be about 10 μg / kg of body weight per day, taking into account the administration route, symptoms, etc. Up to about 1 mg / kg body weight.   The substances of the present invention can be used in their natural form or modified with chemical derivatives Can be formed. As used herein, a molecule is usually part of a molecule. This molecule has another chemical moiety when it has no additional chemical moieties. It is said to be a "conductor." Such moieties are responsible for molecular solubility, absorption, and biological halving. Period can be improved. These moieties alternatively reduce the toxicity of the molecule. May eliminate or attenuate unwanted side effects of the molecule. The part that can mediate such effects is Remington's Pharmaceutical Sciences. (Remington's Pharmaceutical Sciences) (1980).   For example, the immunological characteristics of a functional derivative, such as altered affinity for a given antibody Activation is measured by a competitive type immunoassay. Appropriate changes in immunomodulatory activity Measured by a simple assay. Redox or thermal stability, biological half-life, Hydrophobic, susceptible to proteolysis or aggregate with carriers or multimer Modification of protein properties such as the tendency to aggregate is well known to those of ordinary skill. Assayed in known manner.   The therapeutic effect of the medicament of the present invention may be determined by any suitable means (ie, inhalation, intravenous, intramuscular). , Subcutaneous, intestinal or non-light mouth) to provide medicinal products to patients Can be. To achieve an effective concentration in the blood or tissue in which the growth of the organism is to be controlled Preferably, the medicament of the present invention is administered.   The preferred method to achieve effective blood concentrations is to administer the drug by injection Is Rukoto. Administration can be by continuous infusion or by single or multiple injections .   When providing one of the pharmaceutical products of the present invention to a patient, the dosage of the pharmaceutical product to be administered is: The patient's age, weight, height, gender, general medical condition, previous medical history, etc. Will vary according to various factors. Generally, less or more administration Dose can be administered, but within the range of about 1 pg / kg to 10 mg / kg (patient weight). It is desirable to provide the recipient with a dosage of the pharmaceutical. A therapeutically effective dose is This can be reduced by using the medicament of the present invention or a combination of other medicinal products.   When used in the present invention, either (1) the physiological effect of each compound or (2) each compound Two or more serum concentrations can be measured simultaneously More compounds or medicaments are said to be administered "in combination" with one another. Departure The clear composition should be administered at the same time as, before, or after the administration of other drugs. Can be.   The medicament of the invention reduces the growth rate of the target organism (as identified above) It is intended to be provided to a recipient subject in an amount sufficient for   Administration of the medicament (s) of the invention is either “prophylactic” or “therapeutic”. It can be aimed at fraud. When providing prophylactically, medicines (single or Is provided prior to any symptoms indicative of biological growth. Pharmaceutical (single or Prophylactic administration of multiple) helps prevent, attenuate or reduce the rate of subsequent infection. Treatment When offered as a product, the medicament (s) may be given at the onset of (or immediately Later). Therapeutic administration of the compound (s) may reduce the pathological symptoms of the infection. Helps weaken and increase recovery speed.   The medicament of the present invention can be administered in a pharmaceutically acceptable form and at a therapeutically effective temperature. Administered to animals. If the composition is tolerated by the recipient patient, It is said to be "pharmaceutically acceptable." In such drugs, the dose administered is physiological If they are clinically significant, they are said to be administered in a "therapeutically effective amount." Pharmaceutical existence If the presence results in a detectable change in the physiology of the recipient patient, It is physically significant.   The medicament of the present invention is formulated according to known methods for preparing pharmaceutically useful compositions. These materials or their functions depending on the method The active derivatives are combined in admixture with a pharmaceutically acceptable carrier medium. Other human Suitable vehicles and their formulations, including proteins such as human serum albumin For example, Remington's Pharmaceutical Sciences (Remington's Ph. armaceutical Sciences) (16th edition, Osol, A., Editing, Mack, Pennsylvania Easton, A. (1980)). Pharmaceutical permission suitable for effective administration Such compositions are effective in forming one or more effective amounts to form a tolerable composition. It will contain more of the medicament of the invention together with a suitable amount of carrier medium.   Additional duration of action can be controlled using additional pharmaceutical methods. Controlled release formulation Complexes or absorbs one or more pharmaceutical agents of the present invention Can be achieved using a polymer. Controlling delivery is the right macromolecule (For example, polyester, polyamino acid, polyvinylpyrrolidone, ethylene vinyl Ruacetate, methylcellulose, carboxymethylcellulose or prota sulfate Min) and the method of introduction to control the temperature and release of macromolecules Can be implemented. Controls the duration of action by controlled release formulations Another conceivable method is to convert the pharmaceuticals of the invention into polyesters, polyamino acids, Poly such as drogel, poly (lactic acid) or ethylene vinyl acetate copolymer Into the particles of the mer material. Alternatively, these drugs can be Instead of being introduced into the cell, these materials are applied, for example, by coacervation technology or Microcapsules prepared by interfacial polymerization, e.g. Roxymethylcellulose or gelatin microcapsules and poly (methylmethac Related) in microcapsules or in colloid drug delivery systems such as Riboso , Albumin microspheres, microemulsions, nanoparticles and nanocapsules or Can be confined in a chroma emulsion. Such a technology is available at Remington's It is described in Pharmaceutical Sciences (1980).   The present invention further relates to a pharmaceutical composition of the present invention comprising one or more components loaded with one or more components. A pharmaceutical pack or kit comprising one or more containers is provided. Such a content Related to the device (s) is the drug or biological product Notices in the form prescribed by governmental authorities that regulate their manufacture, use or sale; This notice also requires approval by manufacturing, use or marketing authorities for administration to humans. Reflects. In addition, the medicament of the present invention may be used in conjunction with other therapeutic compounds. Can be   6. Shotgun method for megapace DNA sequencing   The present invention further provides for distribution of greater than 1 megabase using a random shotgun method. This is the first indication that the sequence of the columns can be determined. Detailed description in the following examples This method was used to determine whether overlapping or pre-sequencing The preliminary costs of isolating and sequencing consecutive subclones were eliminated.   The following examples further illustrate certain features of the present invention, and are not intended to be limiting. Is not limited to this.                                  Example Experimental design and method   1. Shotgun sequencing strategy   The overall strategy of the shotgun method for whole genome sequencing is outlined in Table 3. You. The theory of shotgun sequencing is based on the Poisson distribution equation p_x= M^xe^-m/ x! ( Where x is the number of event occurrences, m is the average number of occurrences, and p_xIs a fixed amount The probability that a given base will not be sequenced after a random sequence has occurred) Lander and Waterman (Lander and Waterman, GenomicsTwo: 231 (1988)). L is the length of the genome, n is the number of cloned inserts sequenced and w is the sequencing read If the length is m = nw / L, then the clone is preceded by a fixed base. Probability of not occurring at any of the w bases to run, ie, the probability that the base will not be sequenced Is p₀= E^-mIt is. Using the folding range as the unit for m gives a distribution of 1.8 Mb. After random generation of the columns, it can be seen that m = 1, representing a 1 × range. In this case, p₀= E^-1= 0.37, so approximately 37% are not sequenced . For example, a 5X range (from both the insertion end and the average sequence read length of 460 bp) The sequence of approximately 9500 clones is determined)₀= E^-Five= 0.0067 or 0.67% Are determined. all The gap length of the body is Le^-mAnd the average gap size is L / n. 5X range The box is 128 bp on average, leaving 128 gaps. This process is a And Waterman (GenomicsTwo: 231 (1988)). Table 4 Indicates a range on a 1.9 Mb genome with an average fragment size of 460 bp.   2. Random library construction   To estimate the random model described above during actual sequencing, Nearly ideal models of nome fragments are needed. How to build the next library The law was developed to achieve this.   H. influenza Rd KW20 DNA was prepared by phenol extraction. 60 0 μg DNA, 300 mM sodium acetate, 10 mM Tris-HCl, 1 mM Na-EDT A, A mixture containing 30% glycerin (3.3 ml) was prepared using a 3 mm probe. Sonicated with the lowest energy set at 0 ° C. per minute (Branson Model 450 So nicator). The DNA was ethanol precipitated and redissolved in 500 μl of TE buffer. Was. To create blunt ends, add 100 μl aliquot to 5 units of BAL31 nucleus 10 minutes at 30 ° C. in 200 μl of BAL31 buffer with ze (New England BioLabs) Digested. DNA was extracted with phenol, precipitated with ethanol, and added to 100 μl of TE buffer Redissolve, run on a 1.0% low melting agarose gel, and run 1.6-2.0 k A fraction of size b is cut off, phenol extracted and TE buffer 20 Redissolved in μl. Using the two-step ligation method,> 99% are single-stranded inserts 9 A plasmid library with 7% insert was obtained. First binding mixture (5 0 μl) was 2 μg of the DNA fragment, SmaI / BAP pUC18 DNA (Pharmaci a) containing 2 μg and 10 units of T4 ligase (GIBCO / BRL) Incubation was 14 'for 4 hours. Phenol extraction and ethanol After precipitation, the DNA was dissolved in 20 μl of TE buffer and 1.0% low melting agarose gel. Le Electrophoresed above. Insert (i), vector (v), v + i, v + 2i, v + 3i,. . . Of the ethidium bromide-stained linear band identified as Visualize the dendrites with 360 nm UV light and cut out the v + iDNA, And collected in 20 μl of TE. v + iDNA is the v + i linear, 4 dNTPs each  Contains 500 μM and 9 units of T4 polymerase (New England BioLabs) T4 polymerase treatment for 5 minutes at 37 ° C in the reaction mixture (50 µl) under the recommended buffer conditions To the end of the plant. Recovered after phenol extraction and ethanol precipitation The v + i linear was dissolved in 20 μl of TE. The final join to make a circle is v + i Performed overnight at 14 'in a 50 μl reaction containing 5 μl of linear and 5 units of T4 ligase . After 10 minutes at 70 ', the reaction mixture was stored at -20'.   By this two-step method, the double insertion chimera (<1%) or the free vector (<3 %) To molecularly random collection of single-inserted plasmid recombinants with minimal contamination A collection was obtained. Deviations from randomness are most likely to occur during cloning E. coli host cells lacking all recombination and restriction functions (A. Greener , StrategiesThree(1): 5 (1990)). Prevented loan loss. Spread the transformed cells directly on the antibiotic diffusion plate, The normal broth recovery phase allows for the growth and selection of the fastest growing cells. Escaped. Seeding was carried out as follows:   Epicurian coli SURE II supercompetent cells (Stratagene  200152) was thawed on ice and chilled Falcon (Falc) on ice on) Transferred to 2059 tubes. 1.7 μl of a 1.4 M β-mercaptoethanol fraction was added to the cells. A final concentration of 25 mM was added in addition to the others. Cells were incubated on ice for 10 minutes . Add 1 μl of the final conjugate fraction to the cells and incubate on ice for 30 minutes. I did it. Cells were pulse heated at 42 'for 30 seconds and returned on ice for 2 minutes. any To minimize the preferential growth of certain transformed cells, Growth periods in body culture were excluded from this protocol. Instead, transform The body was treated with SOB agar (1.5% SOB agar: 20 g of tryptone, 5 g of yeast extract, Na A nutrient-rich SOB containing a bottom layer of 0.5 g of Cl, 1.5% Difco Agar / L) Plated directly on the plate. 0.4 ml of Ambicillin (50 mg / ml) / S in 5 ml of bottom layer Replenish 100 ml of OB agar. X-Gal (2%) 1 ml, M on 15 ml of upper layer of SOB agar gCl_Two(1M) 1 ml and MgSO_FourReplenish 1 ml of / 100 ml of / SOB agar. Upper layer 15 ml was poured just before seeding. Our titer is approximately 100 colonies / transformation fractionation Was 10 μl.   All colonies, regardless of size, were picked for mold production. From the library Only clones lost by "toxic" DNA or deleterious gene products are deleted, The number of gaps will increase only slightly more than expected.   H. M13-21 primer to evaluate influenza library quality Was used to obtain sequence data from approximately 4000 templates. 1300, 1800, 2500, 32 After obtaining the 00 and 3800 sequence fragments, the random sequence fragments were: Auto Assembler (registered trademark) software (Perkin-Elmer (AB) A Assembled using pplied Biosystems department) and assembled specific salt The number of base pairs was determined. Based on the above formula, 2.5X10⁶And 1.9X10⁶bp genome As a function of the number of sequence fragments obtained with an average read length of 460 bp An ideal plot of the number of unsequenced base pairs was determined (FIG. 3). 38 Uses actual data from assemblies of up to 00 sequence fragments And plots the progress of the assembly, and is provided with an ideal plot And compare it with the existing data (Figure 3). Figure 3 shows that actual assembly data is ideal That the plot did not essentially deviate from the Ideal with no contamination by the input chimera and no vector Approximate to a random library It shows that it was built.   3. Random DNA sequencing   High quality double-stranded DNA plasmid templates (19,687) Chick Technology Corporation (Gaithersburg, MD) Manufactured using a jointly developed "boiling bead" method (Adams et al., Sciencetwenty five Two : 1651 (1991); Adams et al., Nature.355: 632 (1992)). Preparation of plasmid Perform all DNA preparation steps from bacterial growth to final DNA purification in a 96-well format. gave. The template concentration was Hoechst Dye and Millipore cytofluo. (Millipore Cytofluor). Do not adjust DNA concentration However, where possible, low producing templates were identified and the sequence was not determined. The mold is 2 Also prepared from two H. influenza lambda genomic libraries. Amplification live The rally was constructed in the vector lambda GEM-12 (Promega) and unamplified Bullies were constructed in lambda DASH II (Stratagene). Especially non-amplified lambda For the library, H. influenza Rd KW20 DNA (> 100 kb) , 50 μg of DNA, 1 × Sau3AI buffer, reaction mixture containing 20 units of Sau3AI (200 μl) was partially digested in 23 ′ for 6 minutes. Digested DNA is extracted with phenol And electrophoresed on a 0.5% low melting agar gel at 2 V / cm for 7 hours. Fifteen Fragments of up to 25 kb were excised and recovered in a final volume of 6 μl . 1 μl of the fragment was used in the recommended ligation reaction with the DASHII vector ne) Used with 1 μl. Gigapack II XL packaging Package after recommended protocol with extract (Stratagene, # 227711) 1 μl of the binding mixture was used per zing reaction. The phage is mixed with the packaging The mixture was directly spread without amplification (dilution with 500 μl of recommended SM buffer and After chloroform treatment). Yield about 2.5 × 10^Three pfu / μl. Amplification library Is Essentially as described above, except that the lambda GEM-12 vector was used. Created. After packaging, about 3.5 × 10^Four Spread pfu to restricted NM539 host . Lysate is taken up in 2 ml of SM buffer and frozen in 7% dimethyl sulfoxide Saved. This phage titer is approximately 1 × 10⁹pfu / ml.   Liquid lysates (10 ml) were prepared from randomly selected plaques and the template was Prepared on an anion exchange resin (Qiagen). The sequencing reaction is M13 forward (M1 3-21) and M13 reverse (M13RP1) primers (Adams et al., Nature368: 474 ( Applied Biosystems PRISM Ready Reaction for 1994)) Dye Primer Cycle Sequencing Kit (Ready Reaction Dye AB Catalyst Love Station (Lab) using Primer Cycle Sequencing Kits Station) on the plasmid template. Dye determinator arrangement The constant reaction is Applied Biosystems Ready Reaction Die Termine. Perkin-Elmer 9600 using the Data Cycle Sequencing Kit Performed on a lambda mold in a thermocycler. Using T7 and SP6 primers Sequence the ends of the insert obtained from the Lambda GEM-12 library, and Obtained from lambda DASH II library using T7 and T3 primers The ends of the insert were sequenced. AB373 DNA sequencer per day Three months of sequencing reactions (28,643) were performed by eight people using an average of 14 pieces. Sequencing All reactions use the stretch modification of AB 373 and are primarily 34 cm Analyzed using reading distance. Overall sequencing success rate for M13-21 sequences 84% for the M13RP1 sequence and 83% for the dye terminator reaction. Was 65%. Average usable read length is 485 bp for M13-21 sequence And 444 bp for the M13RP1 sequence and the dye terminator reaction Was 375 bp. Table 5 summarizes the high throughput sequencing phases of the present invention. Things.   Richards, et al. (Richards, et al., Automated DNA sequencing a nd Analysis, MD. Adams, C.I. Fields, J.C. Venter, editing (Academic  Press, London, 1994), Chapter 28), is a show of lambda and cosmid clones. The ends of a sequencing template to facilitate serial alignment in the Tugun assembly project It describes the value of using sequences obtained from the ends. We are M13-21 (previous Orientation) In a sequencing reaction performed with M13RP1 (reverse orientation) compared to the primer. Desirability of double-ended sequencing for shorter read lengths (due to reduced template counts) Including cost reductions). Approximately half of the template is sequenced from both ends Was. In total, 9,297 M13RP1 sequencing reactions were performed. Random reverse alignment Fixed reactions were performed based on successful forward sequencing reactions. Some M13RP One sequence was obtained in a semi-oriented manner: M13-21 pointing outward at the end of the sequence The sequences were specifically selected for M13RP1 sequencing in an effort to align the serials. Half way An assembly strategy is effective, and clone-based alignment is An integral part of the closure was formed (see below).   4. Protocol for automated cycle sequencing   Sequencing was performed with eight ABI Catalyst robots and fourteen AB373. Consisted of using an automatic DNA sequencer. Catalyst robot Is an elaborate, publicly available pipette specially developed for DNA sequencing reactions And temperature control robot. This catalyst is separated from the pre-sorted template, Synonucleotides and dideoxynucleotides, Taq thermostable DNA polymerase Combines a reaction mixture consisting of a fluorescently labeled sequencing primer and a reaction buffer ing. The reaction mixture and mold were transferred to a 96-well aluminum heat cycle plate. Combined together within the le. Denaturation, annealing of primer and template and DNA Linear amplification including synthetic elongation Thirty consecutive cycle steps (eg, one primer synthesis) were performed. Heat circulation pre Heated lid with rubber gasket on plate prevents evaporation and does not require oil overcoating Was.   Two sequencing protocols were used: dye labeled primer and dye labeled dide. Oxy chain terminator. Shotgun sequencing has four terminators -Four dye-labeled sequencing primers, one for each of the nucleotides Is related to the use of Each dye-primer is labeled with a different fluorescent dye And four individual reactions are 373 DNA sequences for electrophoresis, detection and base calling. Can be combined in one lane of the sequencer. AB is currently The mixed reaction mixture is combined with a bulk reagent containing all non-template reagents required for sequencing. Supplied in packages. Generally longer usable due to plasmid template Sequence is obtained, but sequencing can be performed with dye-platin Performed using both PCR generated templates with both imers and dye-terminators Can be applied.   32 reactions per 373 sequencer were performed daily for a total of 960 samples . Electrophoresis was performed at night according to the manufacturer's protocol, and data was collected at 12:00 Gathered for a while. After electrophoresis and fluorescence detection, the AB373 provides automatic lane tracking and Perform base calling. Lane tracking was confirmed visually. D of each array Lectroferogram (or fluorescent lane trace) is visually inspected and quality Was evaluated. Track and retrieve poor quality sequences, and soft Into the Sybase database by software (stored daily on 8 mm tape). The reading vector polylinker sequence is automatically retrieved by a software program. Removed. The average sequence length compiled from standard ABI 373 is around 400 bp and It was largely dependent on the quality of the template used for the sequencing reaction. ABI 373 All sequencers are converted to Stretch Liners, And this provides a longer electrophoresis path before fluorescence detection, and as a result The average number of available bases has increased to 500-600 bp. Information science   1. Data management   Numerous information management systems for large-scale sequencing laboratories (labs) have been developed ( Kerlavage et al., Proceedings of the Twenty-Sixth Annual Hawaii Intern ational Conference on System Sciences, IEEE Computer Society Press, Washington DC,585(1993)). Collect and assemble sequence data This system used to use Sybase related data management system Developed and minimized user errors by automating data flow as much as possible Designed to let. The database covers from template preparation to final analysis of the genome. All information gathered during all operations is stored and correlated. AB37 The raw output of the three sequencers is based on the Macintosh platform And the selected data management system is a Unix platform Based on raw data and analysis results with minimal user effort. A large variety of users and client servers that can flow seamlessly through the base It was necessary to design and run the application. Large array pairs A description of the software program used for preparation and management is provided in FIG.   2. assembly   Assembly engine (TIGR Assenbler) rapidly converts thousands of sequence fragments Developed for accurate assembly. AB Auto Assembler (trademark) Of data related to aligned sequence file output of TIGR assembler To provide a graphical interface to electropherograms for collection purposes Modified as (And called TIGR Editor). TIGR editor (editor) Electropherogram files and Unic for the Macintosh platform Synchronization between sequence data in the Splatform Haemophilus influenzae database Has been maintained.   TIGR assembler collects and assembles fragments of the genome simultaneously . Ten^FourTo get the speed needed to assemble more than fragments, The algorithm creates a compilation table of 10 bp oligonucleotide sequences and Created a segment overlap table. The possible duplication of each fragment is Determine if the fragment appears to be in a repeating element. Single seed array fragment Starting with the TIGR assembler, the TIGR assembler is based on the oligonucleotide content. Extend the latest continuum by trying to add the best matching fragment You. The most recent sequence and candidate fragments are available in Smith for optimal gap alignment. ・ Waterman (Smith-Waterman) algorithm (Waterman, MS, Me thods in Enzymology164: 765 (1988)). Most New continuum should be flagged only if strict criteria for the quality of Extended by the client. Compliance criteria include minimum overlap length and maximum non-conformance Includes edge length and minimum fit percentage. These criteria cover a minimal area Automatically degrades by the algorithm and has a possible repetition factor It rises in the area. The possible duplication of each fragment is Determine if the event appears to be in a repeating element. With repeating elements and potentially chimeras Fragments that represent the boundaries of a fragment are often partially mismatched at the aligned ends. Rejected based on fit and removed from the latest continuation. TIGR assembler Uses clone size information along with sequencing from both ends of each template It is designed to be. This allows the arrangement to be obtained from the two ends of the same template. Row fragments are facing each other in the sequence and have a constant base pairing Columns (for a given library, based on known clone size range (Identifiable in each clone). Flu Assembly of 24,304 sequence fragments of Enza has 512 Mb of RAM 30 hours of CPU time using a single SPARCenter 2000 processor Was needed. Approximately 210 serials were obtained in this way. TIGR assemble Due to the high rigor of Ra, the grasta (modified fasta (Person and Lipman, Pr oc. Natl. Acad. Sci. U.S.A.85: 2444 (1988)) The successors were searched for each other. In this way, the data set in 140 continuum Duplicates were also detected that allowed data summarization. Location and location of each fragment in the sequence And extensive information on the consensus sequence itself I put it in   3. Alignment of assembled continuum   After assembly, the relative positions of the 140 serials were not known. these Serials were aligned by asm align. asm align is used to identify adjacent Use multiple relationships to define and align. Using this algorithm And put 140 serials into 42 groups, for a total of 42 physical gaps (of this region) There is no template DNA and 98 sequence gaps (the template can be used to fill the gap) ). Align and fill gaps separated by physical gaps   Developed four integrated strategies to align serial gaps separated by physical gaps did. Design oligonucleotide primers and synthesize from the end of each serial group did. Therefore, these primers may be used in one or more of the strategies outlined below. Use to use with Was able to:   1. A unique "finger" for 72 subsets of the above oligonucleotides Southern analysis was performed to generate "prints". This method uses Labeled oligonucleotides homologous to the terminus can be used to hybridize To form a lid and consequently a similar or identical hybridizing pattern. It was based on the assumption of sharing turns or "fingerprints". Ori The gonucleotides were 50 pmol each of the 20 mer and 250 mCi of [γ-³²P] ATP and T4 Labeled using polynucleotide kinase. The labeled oligonucleotide is And purified using Sephadex G-25 Superfine (Pharmacia). One frequently used cutter (AseI) and five infrequently used cutters Influenza digested with a cutter (BglII, EcoRI, PstI, XbaI and PvuII) In the Southern hybrid analysis of Rd chromosomal DNA of Lactobacillus casei, 107 cpm oligonucleotide Otide was used. DNA obtained from each digest is fractionated on 0.7% agarose gel And Nytran Plus nylon membrane (Schleicher & Schu) ell). Hybridization was performed at 40 'for 16 hours. Non-specific signals To remove, each plot was taken at room temperature and the stringency condition was set to 0.1 × SSC + 0.5% SDS. 洗浄 高 め 高 め. Convert the plot to a phosphor imager (PhosphorIm ager) Expose to cassettes (Molecular Dynamics) for several hours and hybridize The formation patterns were compared visually.   The adjacent contigs identified in this way were targeted for a specific PCR reaction.   2. Peptide bonds are blastx (Altschul et al., J. Mol. Biol.215: 403 (1990)). If the ends of the two sequences matched the same database sequence in an appropriate way Tentatively considered that these two continuum were adjacent to the difference.   3. Two lambda libraries constructed from Haemophilus influenzae genomic DNA Was searched with oligonucleotides designed from the end of the series (Kirkness et al.). , GenomicsTen: 985 (1991)). Next, create a template using positive plaque , And the sequence was determined from each end of the lambda clone insert. These array flags Were searched using grasta against a database of all continuum. same Align two sequences that match the sequences obtained from the opposite ends of the same lambda clone I let you. The lambda clone then fills in sequence gaps between adjacent rapid successions. A template for providing Lambda clones are particularly valuable for decoding repetitive structures There is.   4. Check the alignment of the continuum found by other methods and Standard and long-range (XL) PCR reactions to establish The reaction was performed as follows.   Standard PCR was performed as follows. Each reaction was 37 μl of the mixture; H_TwoO 16. 5 μl, 25 mM MgCl_Two 3 μl, dNTP mix (1.25 mM each dNTP) 8 μl 4.5 μl of I0X PCR core buffer II (Perkin Elmer), Rd of Haemophilus influenzae It contained 25 ng of KW20 genomic DNA. Two appropriate primers (4 μl, 3.2 p mole / μl) was added to each reaction. Hot start at 95 for 5 minutes followed by 75 ' It was carried out. While maintaining, H_TwoO 4.3μl, 10X PCR core buffer II Amplitaq DNA polymerase (Perkin Elmer) 0 in 0.5 μl 0.3 μl was added to each reaction. PCR profile: 94 '/ 45 sec denaturation; 55' / 1 min Annealing; 25 cycles of 72 '/ 3 min extension. All reactions are par Performed in a 96-well format on a Kin Elmer GeneAmp PCR System 9600.   Long range PCR (XL PCR) was performed as follows: Each reaction was mixed 35.2 μl; H_TwoO 12.0 μl, 25 mM Mg (OAc)_Two 2.2 μl, dNTP mix (20 0 μM final concentration) 4 μl, 12.0 μl of 3.3 × PCR buffer, Haemophilus influenzae Rd Contains 25ng of KW20 genomic DNA Was out. Two appropriate primers (5 μl, 3.2 pmole / μl) were added to each reaction. The hot start was performed at 94 'for 1 minute. For each reaction, 3.3X PCR buffer II 2. 2.0 μl of rTth polymerase (4 U / reaction) in 8 μl was added. PCR profile 18 cycles of annealing and extension of 62 '/ 8 minutes, 94' / 15 second denaturation; Followed by 94 '/ 15 sec denaturation; 62' / 8 min (15 sec increase per cycle) annealing 12 cycles of brushing and extension; 72 '/ 10 minutes final extension. All reactions are -Kin Elmer GeneAmp PCR system 9600 performed in 96 well format.   PCR reactions were performed essentially for each physical gap end combination, but adjacent to each other. Combination needed to align tangent continuum and completely fill gaps To reduce the number of PCR reactions, Southern fingerprinting, data Techniques such as base adaptation and large insert clone search were of particular value. Using these strategies on a larger scale for future genomics projects will not be fully genomic. The overall effectiveness of filling the system will be increased. Aligned by each of these techniques The number of physical gaps caused and filled is summarized in Table 5.   Sequence information from the 15-20 kb clone ends fills in gaps and Particularly suitable for unraveling and providing general information on overall genome assembly I have. We also found that some fragments of the H. influenzae genome It was also of interest that some of the high copy plasmids could not be cloned. We considered that soluble lambda clones provided the DNA for these fragments. amplification Lambda library, prepared template and sequence information obtained from each end Approximately 100 random plaques were collected. These sequences are searched for continuum (gr asta), and combine with those appropriate continuations in the database, and the result Lambda clone scaffolds further contribute to the accuracy of genome assembly Provided (Figure 5). In addition to confirming the continuum structure, lambda clones closed 23 physical gaps . Approximately 78% of the genome was covered by lambda clones.   Lambda clones were also useful for elucidating repetitive structures. Antigens identified in the genome The conformation is composed of six ribosomal RNA operons and one repeat of 5,340 bp in length (2 (Copy), but with a single clone from a random insert library It was small enough to be able to. Oligonucleotide probes for each iteration Design from the beginning distinctive side and form a hybrid with lambda library I let it. Positive plaques were identified for each side and obtained from the ends of each clone. Sequence fragments were used to correctly direct repeats in the genome.   The six ribosomal RNA (rRNA) operons of Haemophilus influenzae (16S sub Unit-23S subunit-5S subunit) can be identified and assembled Whether it can be a complex one that seems to have a significant number of repetitive regions Testing our overall strategy for sequencing and assembling the genome Was. Due to the high degree of sequence similarity and the length of the six operons, the entire underlying An assembly method has been obtained which collects all sequences into a few unidentified continuations. In an array A pair of unique sides for each to determine the correct placement of the operon at A partial arrangement was required. No unique side sequence at left (16S rRNA) end I couldn't do that. This region has a ribosomal promoter, and It did not seem possible to clone in the high copy number pUC18 plasmid. However However, a unique sequence could be identified at the right (5S) end. Oligonucleotide Primers are designed from these six side regions and two lambda libraries Used to suggest Lee. For each of the six rRNA operons, At least completely fill in the peron and have unique side sequences at the 16S and 5S ends Also one positive plaque It has been certified. These plaques were used as templates to obtain unique sequences from six rRNA operators. Provided to each of them.   Further arrangements of the overall structure of the assembled circular genome include the enzymes ApaI, SmaI and R A computer generated restriction map based on the sequence assembled for srII Redfield and Lee (Genetic Maps: locus maps of compl ex genomes, S.J. Edited by O'Brien, Cold Spring Harbor Laboratory Pre ss, New York, NY, 1990, 2110) It was obtained by doing. Restriction fragments of maps derived from sequences are physical The size and relative order were consistent with those obtained from the target map (FIG. 5). Edit   AB Auto Assembler (registered trademark) and First Data Finder ( Fast Data Finder^fM) Assemble overlapping 10 kb fragment of serial using hardware Visually edited by standing up. Auto Assembler (registered trademark) Provides a graphic interface to electropherogram data for collection You. Use electropherogram data to determine the most likely base at each position Assigned. If the difference could not be resolved or a clear assignment could not be made In this case, the automatic base call was not changed. Changes in individual sequences are electropherograms Write to a ram file and use the replication protocol (crash) Simultaneous sequence data between the Enza database and electropherogram files Gender (identity?) Was maintained. After editing, before annotating, TIGR Assembler. The assembly was reassembled using.   Potential frameshifts identified during the annotation of the genome are Omitted as a report. These reports include alignment software (praze) Is the most likely city of lost or inserted bases Display of sequence alignments, including combinations of sequences that are predicted to be I will. Obvious frame shifts can occur in sequences that may need further editing. Used to indicate area. Frameshift is an obvious electropherogram No correction was made if the data did not match the frame shift. Frame shift Was edited using a TIGR editor (Editor).   rRNA and other repetitive regions are used to complete the circular genome by TIGR assembler. Hindered assembly. The final assembly of the genome requires multiple runs based on short duplications. Achieved using comb asm splicing succession together. Genome sequence accuracy   The accuracy of the H. influenzae genomic sequence is Because the fungus sequences are very low and most of these sequences are from other species, Difficult to quantify. However, there are three The accuracy parameter exists. First, based on database similarity , Predicted number of apparent frameshifts of Haemophilus influenzae genes is 148 . Some of these apparent frame shifts are, in particular, 49 explicit frame shifts. Is based on a fit to a hypothetical protein from another organism Given that, in our database sequence rather than in our sequence It seems to be. Second, there are 188 bases in the genome that remain ambiguous as N There is (1 / 9,735bp). Combine these two types of "known" errors We can calculate a maximum sequence accuracy of 99.98%. Average range is 6 .5X, and 1% of the genome is 1-fold in range. Gene identification   Predict all coding regions of the Haemophilus influenzae Rd genome; and Genes, tRNAs and rRNAs and other features of DNA sequences (eg, repeats, signatures) Nodal sites, replication, origin sites, nucleotide composition) were attempted. easily Clarity is provided below for some descriptions of sequence features.   The Haemophilus influenzae Rd genome is a circular chromosome of 1,830,121 bp. Overall G / The content of C nucleotides is approximately 38% (A = 31%, C = 19%, G = 19%, T = 31% , IUB = 0.035%). Genomic G / C content looks for global structural features In order to do this we looked at several length windows. In a 5,000 bp window, G / C Is relatively uniform except for the 7-large G / C-rich region and the A / T-rich region. And so on (FIG. 5). The G / C-rich region consists of six rRNA operons and a mysterious Corresponds to the position of the view-like prophage. Co by bacteriophage mu The genes for some proteins that have similarities to the protein being It is located between 1.56 and 1.59 Mbp. This region of the genome is Have a significantly higher G / C content than the average (about 38% of the rest of the genome) About 50% G / C). Yes, regarding the cause or significance of A / T rich areas The intent has not yet been confirmed.   The minimal replication origin (oriC) of E. coli contains one terminal GATC core sequence 9 containing three copies of the 13 base pair repeat and the other terminal TIAT core sequence. A 245 bp region identified by four copies of a base pair repeat. GATC section The position is a methylation target and controlled replication, while the TIAT site defines the binding site for DnaA. Provided, and is the first step in the replication process (edited by Genes V, B. Lewin (Oxford University Press, New York, 1994), Chapters 18-19). The limits are these same The approximately 281 bp sequence (602,483 to 602,764) specified by the core sequence It appears that fluenza Rd specifies the origin of replication. These Cody Nate is a combination of ribosome operons rrnF, rrnE, rrnD and rrnA, rrnB, rrn Exists between C. Re These two groups of bososome operons are transcribed in opposite directions and the origin substitution is Consistent with the polarity of these transcripts. Termination of E. coli replication involves two replication forks Represented by two 23 bp termination sequences located approximately 100 bp on either side of the midpoint. You. Two potential termination sequences that share a 10 bp core sequence with the E. coli termination sequence are Coordinates for Ruenza bacteria are 1,375,949 to 1,375,958 and 1,558,759 to 1,558,768 Was identified. The coordination of these two sets is the proposed influenza It covers approximately 100 kb from the 180 'opposite of the bacterial origin of replication.   Six rRNA operons were identified. Each rRNA operon has three rRNAs Contains subunits and various spacer regions in the following order: 16S subunit Knit-Spacer area-23S subunit-5S subunit. these Are 1539 bp, 2653 bp and 116 bp, respectively. These three G / C content (50%) of ribosome subunits is higher than that of genome . The G / C content of the spacer region (38%) is consistent with the rest of the genome. Three r The nucleotide sequence of the RNA subunit is 10 for all six ribosomal operons. 0% identical. The rRNA operon is a spacer region between the 16S and 23S sequences. Can be classified into two classes. Of the two spacer regions The shorter one is 478 bp in length (rrnB, rrnE and rrnF) and tRNA Contains the Glu gene. The longer spacer is 723 bp in length (rrnA, rrnC and rrnD) and contains the tRNA Ile and tRNA Ala genes . The set of these two spacer regions is also 100% identical for each group of three operons. . tRNA genes are also present at the 16S and 5S ends of the two tRNA operons doing. The tRNA Arg, tRNA His and tRNA Pro genes are rrnE The tRNA Trp and tRNA ASP genes are located at the 16S Located at the 5S end of A.   The predicted coding region of the H. influenzae genome was initially Codon frequency matrix derived from 122 Haemophilus influenzae coding sequences Using GeneGenemark (Borodovsky and McIninch, Computers Chem.17(2): 123 (1993)). Predicted Sequence region (plus 300 bp side sequence) was specially created for annotation Used in searching a database of non-redundant bacterial proteins (NRBP). DNA All coding regions are drawn from Genebank (Release 85) and are of the same species The resulting sequences were searched for each other. > 97 over a region of> 100 nucleotides Sequences with% similarity were combined. In addition, the sequence is translated, and Switzerland Used for protein comparison with all sequences (Release 30) in the plot (Swiss-Prot) Used. Species belonging to the same species and having> 98% similarity over 33 amino acids Combined columns. NRBP consists of 23,751 genes from 1,099 different species 21,445 arrays derived from bank array and 11,183 Swiss plot arrays It is composed of   A total of 1,749 constitutive predictive coding regions were identified. Influenza bacteria prediction The search for the loaded region is performed by reading three additional strands (strands) to search for NRBP. Translates the interrogated DNA sequence in the open reading frame and matches the protein sequence that matches the interrogated sequence. And use praz to align protein-protein matches. The Gorism, Modified Smith-Wateroman (Pearson And Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988)) Al Implemented using the algorithm. Flame shrinkage by insertion or deletion in the DNA sequence If a shift error occurs, the alignment algorithm starts in the region of maximum similarity, And aligned to the same database fit in another frame using the 300 bp side region Was extended. Areas known to contain frame shift errors It was omitted from the database and evaluated for possible modifications. Unidentified prediction The coding region and the rest of the intergenic sequences are as shown in Swiss Plot, PIR and Geneba. A data set of all peptide sequences available from the company was searched. Operon structure Structure identification is facilitated by experimental measurements of transcription promoters and termination sites. Would.   Each inferred Haemophilus influenzae gene was identified by Riley (Riley, M., Microbiology Reviews57(4): 862 (1993)) Assigned to one of the logical role categories. Allocation is based on the prediction coding region Perform the protein sequence in relation to the Swiss plot sequence in the relay database. Was. Of the 1,749 predicted coding regions, 724 have not been assigned a role. these Of these, 384 did not find a database match, while 340 Conformed to the “hypothetical protein” of the authors. Role assignment is 1,02 in the predicted code area Made in five. Editing of predicted coding regions, their unique identifiers, 3-letter genes The identifier, percent identity, percent similarity and amino acid match length are reported in Table 1 ( a).   Complete genome maps annotated with Haemophilus influenzae Rd are shown in FIGS. ). This map places each predicted coding region on the H. influenzae chromosome. , Indicates its transfer direction, and the color is encrypting its role assignment. Role assignment The assignment is also shown in FIG.   Investigation of the gene and gene chromosome mechanism of Haemophilus influenzae Rd The explanation becomes possible. Haemophilus influenzae survives as a free living organism, Nutritional requirements for growth in the laboratory and especially its pathogenicity and virulence It needs features that make it different from other organisms as related. Genome is life With the complete complement of a class of genes known to be essential Would be expected. For example, the potential phase of the Haemophilus influenzae database The published protein sequences of the homolog and the E. coli ribosome correspond one-to-one. Similarly, as shown in Table 1 (a), aminoacyl-tRNA synthetase Amino acids exist in the genome Are there. Finally, the location of the tRNA gene was mapped on the genome. Typical There are 54 identified tRNA genes including 20 amino acids.   Haemophilus influenzae is fermented and / or electronized to survive as a free living organism Energy must be generated in the form of ATP by transport. Accidental dislike As an aerobic organism, Rd of Haemophilus influenzae is glucose, fructose, galacto. It is known to ferment glucose, ribose, xylose and fucose (Doro cicz et al. Bacteriol.175: 7142 (1993)). Genes identified in Table 1 (a) Is a phosphoenolpyruvate-phosphotransferase system (PTS) and Demonstrating that a transport system can be used for the uptake of these saccharides by the PTS mechanism I have. PTS-based common phosphate carrier enzymes I and Hpr (ptsI and ptsH) The specific gene as well as the glucose-specific crr gene were identified. ptsI, ptsH And the crr gene constitutes the pts operon. However, we have membrane bound glucose No specific enzyme II has been identified. The latter enzyme is necessary for glucose transport in the PTS system. It is important. A complete PTS line of fructose was identified.   Genes encoding a complete glycolysis system and production of fermentation end products have been identified. Increase Reproductive anaerobic respiration mechanisms include nitrate, nitrite and dimethyl sulfoxide. Genes encoding functional electron transport systems using such inorganic electron acceptors Was found by determining Three enzymes of the tricarboxylic acid (TCA) cycle Does not appear to be in the genome. Citrate synthase , Isocitrate dehydrogenase and accordase (acordtase? Acid hydratase) either searches for the predicted coding region or reverses the E. coli enzyme. Not found when used as peptide query for whole genome in translation . This corresponds to the very high glutamate values required in the specified culture medium (1 g / L) (Klein and Luginbuhl, J. Gen. Microbiol.113 : 409 (1979). Glutamate is exchanged for glutamate dehydrogenase. By converting it to faker glutarate, it can be directed to the TCA circuit. You. In the absence of a complete TCA cycle, glutamate probably diverges from the TCA cycle Serving as a carbon source for amino acid biosynthesis by using a precursor One. Functional electron transport systems produce ATP using oxygen as the final electron acceptor Can be used for   Unanswered questions about pathogenicity and virulence are adhesion and adhesion. By testing for certain classes of genes, such as the lipooligosaccharide-generating genes, Can be handled. Moxon and co-workers (Weiser et al., Cell)Five 9 : 657 (1987)) is a tetramer repeat in which these virulence-related genes are arranged vertically. And this repeat is frequently repeated during replication by one or more repeat units. Additional additions and deletions, resulting in an altered reading frame for this gene and Has evidence that its expression has been altered. Now the complete genome The array is used to determine the location of all such array areas (FIG. 5). To determine their role in the phase variation of such potential virulence genes It is possible to start.   Haemophilus influenzae Rd has a very efficient natural DNA transformation system (Kahn and Smith, J. Membrane Biol.138: 155 (1984). Unique DNA collection Sequence site, 5 'AAGTGCGG present in multiple copies in the genome T has been shown to be required for efficient DNA uptake. Now all of these It is possible to determine the location of all sites and to fully describe their distribution. Fifteen genes involved in transformation have been described and sequenced. (Redfield, R., J. Bacteriol.173: 5612 (1991); Chandler, M .; Proc. Natl. Acad. Sci. U.S.A.89: 1616 (1992); Barouki and Smi th, J. Bacteriol.163(2) : 629 (1985); Tomb et al., Gene104: 1 (1991); Tomb, J., Proc. Natl. Acad. Sci. U.S.A.89: 10252 (1992)). 6 genes, comA to comF Is a 22 bp balindrome receptor around one helical turn upstream of the promoter. Including operons that are explicitly (?) Controlled by the capacity regulating element (CRE) I have. The rec-2 transforming gene is also controlled by this element. Now, the genome The location of additional copies of the CRE within and potential transformation under CRE control It is possible to discover genes. In addition, a wide range of other adjustment elements are now easily It is possible to discover, which was not possible before.   One well-described gene regulatory system in bacteria is that certain environmental signals Molecule that detects DNA and a regulatory molecule that is phosphorylated in the activated form of the sensor Is a "two-component" system composed of Regulatory proteins are generally Therefore, a transcription factor that starts or stops a specific gene set when activated ( For a review, see Albright et al., Ann. Rev. Genet.twenty three; 311 (1989); Parkin son and Kofoid, Ann. Rev. Genet.26: 71 (1992)). E. coli is 40 cents It is believed to have a sir-regulatory pair (Albright et al., Ann. Rev. Gene). t.twenty three: 311 (1989); Parkinson and Kofoid, Ann. Rev. Genet.26: 71 (1992 Year)). The Haemophilus influenzae genome was analyzed using tblastn and tfasta, Using the protein, we searched from sensor and regulatory protein families. Four sensors And five regulatory proteins have been identified and are similar to proteins from other species. (Table 6). Except for CpxR, there is a corresponding sensor for each regulatory protein. Seems to be. By searching for CpxA protein obtained from E. coli, Thus, three of the four sensors shown in Table 6 were identified, with additional significant No fit was seen. The level of sequence similarity is sufficient to be undetectable by tfasta May be low. Representative of NtrC class regulatory proteins Was not seen at all. This class of proteins is a signal of RNA polymerase Interacts directly with the subunit 54, which is present in H. influenzae Not. All regulatory proteins fall into the OmpR subclass (Albright et al., Ann . Rev. Genet.twenty three: 311 (1989); Parkinson and Kofoid, Ann. Rev. Gene t.26: 71 (1992)). Haemophilus influenzae phoBR and basRS genes are next to each other Are in contact and possibly form an operon. The nar and arc genes are adjacent to each other Not located.   The questions of most interest that can be answered by a complete genome sequence are: It is about missing genes or pathways. Nonpathogenic influenza R The d strain is significantly different from the pathogenic serotype b strain. Many differences between these two strains Or appear to be factors affecting infectivity. For example, bacteria and host cells Gene group with cilia involved in adhesion with (Mol. Microbiol.13: 673 (1994)) is now absent in the Rd strain. It is shown. Influenza type b strains have ciliated populations on their sides The pepN and purE genes are adjacent to each other in the Rd strain (FIG. 7), and This suggests that the entire group (duster → cluster) has been clipped. Than At a broad level, a nonredundant set of protein-encoding genes from E. coli Ie, the Genome Project at the University of Wisconsin Genebank. ject) Serial: Genebank acceptance D10483, L10328, U00006, U00039, U140 03 and U118997, 1,216 predicted protein sequences (Yura et al., Nucleic  Acids Research20: 3305 (1992); Burland et al., Genomics.16: 551 (1993 Utilizing)), we can see which proteins in E. coli are present in H. influenzae Not decided. The minimum threshold of a match is scored as positive, even for weak matches, And thereby the minimum estimate of E. coli genes not found in H. influenzae Gave. E. coli protein using tblastn Each of the qualities was searched against the complete genome. A blast score> 100 was considered a match. Combination A total of 627 E. coli proteins represent at least one region of the H. influenzae genome And 589 proteins did not match. 589 non-conforming proteins Quality and that they contain an unbalanced number of hypothetical E. coli proteins. I understood. 68% of E. coli proteins identified Matched by sequence, while only 38% of the hypothetical protein matched. Ta Protein is hypothetical based on its incompatibility with any other known protein. Annotate as protein (Yura et al., Nucleic Acids Research)20: 3395 ( 1992); Burland et al., Genomics16: 551 (1993)). Incompatible proteins There are at least two possibilities for over-presentation of hypothetical proteins. Can provide a compelling explanation: hypothetical proteins are actually translated Not (at least in the annotated frames) these are E. coli-specific Protein, and most closely related species except for Salmonella typhimurium It doesn't seem to be found in any species.   A total of 384 predicted coding regions are available with 6-frame translations of GeneBank Release 87 Did not show similarity. These unidentified coding regions encode fasta And compared with each other. Several new gene clusters have been identified. For example, data The two predicted coding regions (HI0591, HI0852) that do not match the base Nearly full length (139 and 143 amino acid residues, respectively) shares 75% identity Have. These areas are similar to each other but are available in the current database The incompatibility with any protein indicates that they may represent a novel cellular function. And suggests.   Unidentified coding regions may have no significant amino acid identity. Membrane potential often retained between receptor members and transport genes Shows the pattern of the panning domain Other types of analysis can be applied, including hydrotherapy. Membrane binding Chan Potential transmembrane with a periodic pattern characteristic of Nenru protein Figure 5 shows five examples of unidentified predicted coding regions showing the rain domain FIG. Using such information, targeted deletions or bursts of these genes Focus on specific features of cell function affected by mutation Wear.   Interest in the medically important features of Haemophilus influenzae biology is It is particularly focused on genes that determine the traits of the disease. Characteristics of bacteria and catalase genes The signature is determined and sequenced as a potential virulence-related gene. (Bishai et al., J. Bacteriol.176: 2914 (1994)). The cause of capsular polysaccharides A number of genes have been mapped and sequenced (Kroll et al., Mol. Microb. iol.Five(6): 1549 (1991)). Several outer membrane protein genes have been identified and (Langford et al., J. Gen. Microbiol.138: 155 (1992)) . The genes for the lipooligosaccharide component of the outer membrane and its synthetic pathway have been intensively studied. (Weiser et al., J. Bacteriol.173: 3304 (1990). Vaccines are available However, studies of outer membrane components have been somewhat motivated by the need to improve vaccines. ing. Data availability   The Haemophilus influenzae genome sequence is available under the accession number L42023 in the Genome Sequence Database ( Genome Sequence Data Base (GSDB). Identified open Nucleotide sequence and peptide of each predicted coding region with start and stop codons Translations have also been accepted by the GSDB. Production of antibodies against Haemophilus influenzae proteins   A substantially pure protein or polypeptide is transfected Or from transformed cells using any one of the methods known in the art. Isolated. Proteins can also be produced in recombinant prokaryotic expression systems, such as E. coli. Or can be chemically synthesized. Tan in final preparation The concentration of the protein is, for example, concentrated by an Amicon filter device. Adjusted to a level of a few micrograms / ml. Next, for this protein Monoclonal or polyclonal antibodies can be prepared as follows. RU:             Production of monoclonal antibodies by hybridoma fusion   Any epitope of the peptide identified and isolated as described Monoclonal antibodies are available from Kohler, G. and Millstein. Milstein C. (Naturetwenty five: 495 (1975)) or the classical method Can be prepared from a murine hybridoma. Simply saying For example, mice can be used for a few weeks for a few micrograms of the selected protein. Inoculate repeatedly. The mice are then sacrificed and spleen antibody-producing cells are isolated. Let go. Splenocytes are fused with mouse myeloma cells by polyethylene glycol And excess non-fused cells were isolated on selective medium containing aminobuterin (H The system is grown and destroyed in AT medium). Dilute the successfully fused cells and dilute The another is placed in a microtiter well where the growth of the culture is continued. Antibody producing claw Engval, E. (Meth. Enzymol.70: 419 (1980 Immunoassays such as the Eliza method and its modification, first described by The antibody in the supernatant of the well is detected and identified by the method. Selected positive claw To collect and use those monoclonal antibody products be able to. Detailed methods for producing monoclonal antibodies are provided by Davids L. L.) etc. (Basic Methods in Molecular Biology Elsevier, New York, Chapter 21-2 (1989).                    Polyclonal antibody production by immunoassay   A polyclonal antiserum containing a heterologous epitope of the mono-protein An expressed protein as described above, which can be absent or modified to increase immunogenicity By immunizing an appropriate animal. Effective poly Clonal antibody production is affected by a number of factors associated with both plateaus and host species. box office. For example, small molecules tend to be less immunogenic than other molecules, and It may require the use of carriers or adjuvants. In addition, the host animal is Fluctuating in response to dose and dose, and inappropriate or excessive antigen can get. Small amounts (ng level) of antigen administered to multiple intradermal sites are the most reliable Seems to be. Efficient immunization protocol for rabbits Vaitukaitis, J. et al. (J. Clin. Metab.33: 988〜991 (1978 ) Can be seen.   Give booster injections at regular intervals and semi-quantitative methods, e.g., of known concentration Antiserum antibody titer as determined by double immunodiffusion in agar against antigen When serum begins to fall, antiserum can be collected. For example, Ouchitaroni -Ouchterlony, O, etc. (Handbook of Experimental Immunology, See Wier D, Editing, Chapter 19 in Blackwell (1973). Plateau concentration of antibody is usually 0 It is in the range of .1 to 0.2 mg / ml serum (about 12 μM). Antiserum affinity for antigen Are, for example, Fisher D. (Manual of Clinical Immn) ology, 2nd edition, edited by Rose and Friedman, Amer. Soc. For Microbiology, Washington DC Competition binding curves as described in (42) in (1980) Create and measure.   Antibody preparations prepared according to either protocol Useful in quantitative immunoassays that measure the concentration of substances with antigen in a sample They can also be used semi-quantitatively or to identify the presence of an antigen in a biological sample. Also used qualitatively. Preparation of PCR primers and amplification of DNA   Various plasmids of the Rd genome of Haemophilus influenzae as disclosed in Tables 1 (a) and 2 According to the present invention to create PCR primers for a variety of applications. Can be used. The PCR primer is preferably at least 15 bases, and And more preferably at least 18 bases in length. Primer sequence When primers are selected, the primer pairs should have approximately the same G so that the melting points are approximately the same. It preferably has a / C ratio. PCR primers and amplified DN of this example A has uses in the following examples. Gene expression from DNA sequence corresponding to ORF   The fragments of the R. flu genome provided in Table 1 (a) or 2 are: It is introduced into an expression vector using conventional techniques. (The cloned sequence is Expression vectors directing protein translation in mammalian, yeast, insect or bacterial expression systems Transfer techniques are well known in the art. ) Commercially available vectors and Expression systems include Stratagene (La Jolla, CA), Promega (Madison, WI) and Invitrogen (Inv) from a variety of suppliers, including itrogen (San Diego, CA) be able to. If desired, enhance expression and fold the appropriate protein To facilitate this, US Pat. No. 5,082,767 to Hatfield et al. This is incorporated herein by reference) and the sequence Codon association and codon pairing can be optimized for a particular expression organism.   The following is the cloned ORF of the genomic fragment of Haemophilus It is provided as one exemplary method of generating the peptide (s). This O Since RF is of bacterial origin and lacks the polyA sequence, this sequence may be, for example, The polyA sequence was added and the BglI and SalI restriction endonuclease enzymes were used. Vector pXT1 (Stratagene) for use in eukaryotic expression systems And spliced from pSG5 (Stratagene) to construct Wear. pTX1 is the gag gene obtained from LTR and Moroni-mouse leukemia virus 1 part of Stable transfection depending on the position of the LTR in the construct Is possible. The vector contains the herpes simplex thymidine kinase promoter and And a selective neomycin gene. Haemophilus DNA is Bacteria using oligonucleotide primers that are complementary to DNA of the genus PstI obtained by PCR from the vector and introduced into the 5 ′ primer For restriction and BglII at the 5 'end of the corresponding Haemophilus DNA 3' primer It has an endonuclease sequence and ensures that the DNA of Haemophilus Care has been taken to follow with the array. Obtained from the resulting PCR reaction The purified fragment was digested with PstI and blunt-ended with exonuclease. And digested with BglII, purified and ligated with pTX1, now polyA and digested. Contains BglII.   The bound product is lipofectin (Lipofin) under the conditions outlined in the product specification. ectin) (Life Technologies, Inc., Grand Island, NY) To transfect into mouse NIH 3T3 cells. Positive tiger The transfection product was obtained by transfecting the cells with 600 μg / ml of G418. (Sigma, St. Louis, MO). Protein The quality is preferably released into the supernatant. However, proteins are not membrane-bound Proteins are retained in the cell or Is restricted to cell surface expression.   As the transfection product must be purified and Mice were injected with a synthetic 15-mer peptide synthesized from the Haemophilus DNA sequence. Antibodies against polypeptides encoded by Haemophilus genus DNA Generate.   If antibody production is not possible, add the Haemophilus DNA sequence into a eukaryotic expression vector. In addition, they are introduced and expressed, for example, as chimeras with β-globin. β-g Antibodies to robin are used to purify the chimera. Next, β-globin Using the corresponding protease cleavage site processed between the gene and Haemophilus DNA After translation, the two polypeptide fragments are separated from each other. β-glo One useful expression vector for generating bin chimerics is pSG5 (Strat agene). This vector encodes rabbit β-globin. Rabbit β -Globin gene intron II promotes splicing of expressed transcripts And the polyadenylation signal introduced into this construct enhances expression values. These techniques described are well known to those skilled in the field of molecular biology. Standard one The law has been published in method textbooks such as Davis (previously described) and others. And Stratagene, Life Technologies, Inc. (Life Technologies, Inc.) ) Or obtain a number of methods from Promega technical support representatives it can. The polypeptide can be purchased from the in vitro express translation kit. (Express^TM In vitro translation such as Translation Kit) (Stratagene) The system can be used to additionally produce from either construct.   Although the present invention has been described in some detail for purposes of clarity and understanding, Various changes in form and details may depart from the true scope of the invention. You will understand what can be done.   All patents, patent applications and publications mentioned above are hereby incorporated by reference. Include in the description. Fatty acid / phospholipid metabolism Acetyl coenzyme A acetyltransferase (thiolase) (fadA) {cross Tridium acetobutylicum} FadR protein (fadR) involved in fatty acid metabolism {E. coli} (3R) -Hydroxymyristol acyl carrier protein dehydrase (fabZ) {large Enterobacteria} 3-ketoacyl-acyl carrier protein reductase (fabG) {E. coli} Acetyl-CoA carboxylase (accA) {E. coli} Acyl carrier protein (acpP) {E. coli} Acyl-CoA thioesterase II (tesB) {E. coli} Beta-ketoacyl-ACP synthase (fabB) {E. coli} Beta-ketoacyl-acyl carrier protein synthase III (fabH) {E. coli} Biotin carboxyl carrier protein (accB) {E. coli} Biotin carboxylase (accC) {E. coli} D-3-hydroxydecanoyl- (acyl carrier protein) dehydratase (fabA) {E. coli} Diacylglycerol kinase (dgkA) {E. coli} Long chain fatty acid coA ligase {Homo sapiens} Malonyl coenzyme A-acyl carrier protein transacylase (fabD enzyme {E. coli } Short-chain alcohol dehydrogenase homolog (envM) {E. coli} USG-1 protein (usg) {E. coli} 1-acyl-glycerol-3-phosphate acyltransferase (plsC) { E. coli} CDP-diglyceride synthetase (cdsA) {E. coli} Glyceres-3-phosphate acyltransferase (plsB) {E. coli} Phosphatidylglycerophosphate phosphatase B (pgpB) {E. coli} Phosphatidylglycerophosphate synthase (pgsA) {E. coli} Phosphatidylserine decarboxylase proenzyme (psd) {E. coli} Phosphatidylserine synthase (pssA) {E. coli} Protein D (hpd) {Hemophilus influenzae} Purines, pyrimidines, nucleosides and nucleotides Purine ribonucleic acid Leotide biosynthesis 5'-phosphoribosyl-5-amino-4-imidazole carboxylase II (pu rK) {E. coli} 5'-phosphoribosyl-5-aminoimidazole synthetase (purM) {large intestine Fungus} 5 'guanylate kinase (gmk) {E. coli} Adenylate kinase (ATP-AMP transphosphorylase) (adk) {Hemofu Virus influenza} Adenylosuccinate lyase (purB) {E. coli} Adenylosuccinate synthetase (purA) {E. coli} Amidophosphoribosyltransferase (purF) {E. coli} Formylglycinamide ribonucleotide synthetase (purL) {E. coli} Formyltetrahydrofolate hydrolase (purU) {E. coli} guaA protein (guaA) {E. coli} Inosine-5'-monophosphate dehydrogenase (guaB) {Acinetobacter cal Coacechicus} Nucleoside diphosphate kinase (ndk) {E. coli} Phosphoribosylamine-glycine ligase (purD) {E. coli} Phosphoribosylaminoimidazole carboxylase catalytic subunit (pur E) {Hemophilus influenza} Phosphoribosylaminoimidazolecarboxamide formyltransferase (purH) {E. coli} Phosphoribosylglycinamide formyltransferase (purN) {E. coli} Phosphoribosyl pyrophosphate synthetase (prsA) {S. typhimurium} SAICAR synthetase (purC) {B. pneumoniae} Pyrimidine ribonucleotide biosynthesis Dihydroorotate dehydrogenase (dihydroorotate oxidase) (pyrD) {E. coli} Orotate phosphoribosyltransferase (pyrE) {E. coli} orotidine 5'-monophosphate (OMP) decarboxyla with pyrF operon coding -Sease {E. Coli} pyrF protein (pyrF) {E. coli} Uracil phosphoribosyltransferase (pyrR) {Bacillus caldrich Scum} 2'-deoxyribonucleotide metabolism Anaerobic ribonucleoside-triphosphate reductase (nrdD) {E. coli} Deoxycytidine triphosphate deaminase (dod) {E. coli} Deoxyuridine triphosphatase (dut) {E. coli} Glutaredoxin (grx) {E. coli} nrdB protein (nrdB) {E. coli} Ribonucleoside-diphosphate reductase 1 alpha chain (nrdA) {E. coli} Thioredoxin reductase (trxB) {E. coli} Thymidylate synthetase (thyA) {E. coli} Reuse of nucleosides and nucleotides 2 ', 3'-cyclic-nucleotide 2'-phosphodiesterase (cpdB) {E. coli} Adenine phosphoribosyltransferase (apt) {E. coli} Adenosine-tetraphosphatase (apaH) {E. coli} Cytidine deaminase (cytidine aminohydrolase) (cda) {E. coli} Cytidylate kinase (cmk) {E. coli} Cytidylate kinase (cmk) {E. coli} Purine-nucleoside phosphorylase (deoD) {E. coli} Thymidine kinase (tdk) {E. coli} Uracil phosphoribosyltransferase (upp) {E. coli} Uridine phosphorylase (udp) {E. coli} Xanthine guanine phosphoribosyltransferase gpt (xgprt) {large Enterobacteria} Xanthine-guanine phosphoribosyltransferase (xgprt) {rat Salmonella typhi} Estimated ATPase (mrp) {E. coli} Sugar-nucleotide biosynthesis, conversion 5'-nucleotidase (ushA) {Homo sapiens} CMP-NeuNAc synthetase (siaB) {meningococcus} Galactose-1-phosphate uridyltransferase (galT) {Hemofil Influenza} Glucose phosphate uridyltransferase (galU) {E. coli} udp-glucose 4-epimerase (galactowardenase) (galE) {Hemo Fils Influenza} UDP-N-acetylglucosamine pyrophospholyase (glmU) {E. coli} Nucleotide and nucleoside interconversion Deoxyguanosine triphosphate triphosphohydrolase (dgt) {E. coli} Uridine kinase (uridine monophosphokinase) (udk) {E. coli} Adjustment function Adenylate cyclase (cyaA) {Hemophilus influenzae} Anaerobic respiratory control protein ARCA (DYE resistance protein) (arcA) {E. coli} Anaerobic respiratory control sensory protein (arcB) {E. coli} araC-like transcriptional regulator {Streptomyces lipidans} Arginine repressor protein (argR) {E. coli} arsC protein (arsC) {plasmid R773} ATP-dependent proteinase (lon) {E. coli} ATP: GTP 3'-pyrophosphotransferase (relA) {E. coli} Carbon starved protein (cstA) {E. coli} Carbon storage regulator (csrA) {E. coli} Cyclic AMP receptor protein (crp) {Hemophilus influenzae} Cyclic AMP receptor protein (crp) {Hemophilus influenzae} cys regulon transcriptional activator (cysB) {E. coli} Ferric absorption regulation protein (fur) {E. coli} Ciliary transcriptional regulator (pilB) {gonococci} Ciliary transcriptional regulator (pilB) {gonococci} Horyl polyglutamic acid-hydidrofolate synthetase expression regulator (accD) { E. coli} Fumaric acid (and nitrate) reduction regulatory protein (fnr) {E. coli} Galactose operon inhibitory factor (galS) {Hemophilus influenzae} Glucokinase regulator {Lattus norpegicus} Glycerol-3-Luglone phosphate inhibitor (glpR) {E. coli} Glycerol-3-Luglone phosphate inhibitor (glpR) {E. coli} Glycine cleavage system transcription activator (gcvA) {E. coli} GTP-binding protein (era) {E. coli} GTP-binding protein (obg) {Bacillus subtilis} Hydrogen peroxide-inducible activator (oxyR) {E. coli} L-fucose operon activator (fucR) {E. coli} lacZ expression regulator (icc) {E. coli} Leucine-responsive regulatory protein (lrp) {E. coli} Leucine-responsive regulatory protein (lrp) {E. coli} LEXA inhibitor (lexA) {E. coli} Lipooligosaccharide protein (lex2A) {Hemophilus influenzae} Lipooligosaccharide protein (lex2A) {Hemophilus influenzae} metF apo repressor (metJ) {E. coli} Molybdenum transport system alternate nitrogenase regulator (modD) {Rhodobacter cap Slatas} msbB protein (msbB) {E. coli} msbB protein (msbB) {E. coli} Negative regulator of translation (relB) {E. coli} Negative rpo regulator (mclA) {E. coli} Nitrate sensing factor protein (narQ) {E. coli} Nitrate / nitrite response regulator protein (narP) {E. coli} Nitrogen regulatory protein P-II (glnB) {E. coli} Guanosine pentaphosphate-3'-pyrophosphohydrolase (spoT) {E. coli} Regron phosphate sensing protein (phoR) {E. coli} Regron phosphate transcription regulatory protein (phoB) {E. coli} Putative nadAB transcription regulator (nadR) {E. coli} Purine nucleotide synthesis inhibitor protein (purR) {E. coli} Putative mulein gene regulator (bolA) {E. coli} rbs repressor (rbsR) {E. coli} Regulatory protein (asnC) {E. coli} Regulatory protein sfsl (sfsA) involved in maltose metabolism {E. coli} Suppressor for cytochrome P450 (Bm3R1) Bacteria} RNA polymerase sigma-32 factor (heat shock regulatory protein F334) (rpoH) { E. coli} RNA polymerase sigma-70 factor (rpoD) {E. coli} RNA polymerase sigma-E factor (rpoE) {E. coli} Sensing factor protein for basR (basS) {E. coli} Stringent starvation protein (sspB) {E. coli} Stringent starvation protein A (sspA) {Hemophilus influenzae} trans-activator for metE and metH (m8tR) {Hemophilus Influenza} Transcriptional activator (tenA) {Bacillus subtilis} Transcription activator protein (ilvY) {E. coli} Transcription regulatory protein (basR) {E. coli} Transcription regulatory protein (tyrR) {E. coli} Tryptophan inhibitor (trpR) {Enterobacter aerogenes} uxu operon regulator (uxuR) {E. coli} Xylose operon regulatory protein (xylR) {E. coli} Duplication DNA = replication, restriction / modification, recombination A / G-specific adening lycosylase (mutY) {E. coli} Chromosome replication initiation factor protein (dnaA) {E. coli} Chromosome replication initiation factor protein (dnaA) {E. coli} Cross-linked endodeoxyribonuclease (ruvC) {E. coli} dfp protein (dfp) {E. coli} DNA adenine methylase (dam) {E. coli} DNA gyrase, subunit A (gyrA) {E. coli} DNA gyrase, subunit B (gyrB) {E. coli} DNA helicase II (urvD) {Hemophilus influenzae} DNA ligase (lig) {E. coli} DNA unsuitable protein (mutH) {E. coli} DNA mismatch repair protein (mutS) {E. coli} DNA mismatch repair protein MUTL (mutL) {E. coli} DNA polymerase I (polA) {E. coli} DNA polymerase III beta subunit (dnaN) {E. coli} DNA polymerase III delta prime subunit (holB) {E. coli} DNA polymerase III delta subunit (holA) {E. coli} DNA polymerase III epsilon subunit (dnaQ) {E. coli} DNA polymerase III, alpha chain (dnaE) {E. coli} DNA polymerase III, chi subunit (holC) {Hemophilus inf Luense} DNA polymerase III, psi subunit (holD) {E. coli} DNA primase (dnaG) {E. coli} DNA recombinase (recG) {E. coli} DNA repair protein (recN) {E. coli} DNA topoisomerase I (topA) {Bacillus subtilis} DNA-3-methyladening ricosidase I (tagl) {E. coli} DNA-dependent ATPase, DNA helicase (recQ) {E. coli} dod protein (dod) {Selaysia marcescens} Dose-dependent dnaK inhibitor protein (dksA) {E. coli} Formamidopyrimidine-DNA glycosylase (fpg) {E. coli} Glucose-suppressed split protein (gidA) {E. coli} Glucose-suppressed split protein (gidB) {E. coli} Hin recombination promoting factor binding protein (fis) {E. coli} Hincll endonuclease (Hincll) {Hemophilus influen Ze} Hindll modified methyltransferase (hindllM) {Hemophil Influenza} Hindll restriction endonuclease (hindllR) {Hemophilus i Influenza} Holiday binding DNA helicase (rubA) {E. coli} Holiday binding DNA helicase (ruvB) {E. coli} Integrase / recombinase protein (xerC) {E. coli} Integrating host factor alpha-subunit (himA) {E. coli} Integrative host factor beta subunit (IHF-beta) (himD) {E. coli} Methylated-DNA-protein-cysteine methyltransferase (da tl) {Bacillus subtilis} mioC protein (mloC) {E. coli} Modified methylase HgiDl (MHgiDl) {Herpetosiphon Aulanchiac { Modified methylase Hincl (hinclM) {Hemophilus influenzae} Mutagen factor mutT (AT-GC mutation) {E. coli} Negative regulator of initiation of replication (seqA) {E. coli} Primosomal protein n precursor (priB) {E. coli} Primosomal protein replication factor (priA) {E. coli} Putative ATP-dependent helicase (dinG) {E. coli} recF protein (recF) {E. coli} recO protein (recO) {E. coli} Recombinase (recA) {Hemophilus influenzae} Recombinant protein (rec2) {Hemophilus influenzae} recR protein (recR) {E. coli} Regulatory protein (recX) {Pseudomonas fluorescens} rep helicase (rep) {E. coli} Replication protein (dnaX) {E. coli} Replicating DNA helicase (dnaB) {E. coli} Restriction enzyme (hgiDIR) {Herpetosiphon giganteus} S-adenosylmethionine synthetase 2 (metX) {E. coli} Shaflon-specific DNA recombinase (rci) {E. coli} Single-stranded DNA binding protein (ssb) {Hemophilus influenzae} Site-specific recombinase (rcb) {E. coli} Topoisomerase I (topA) {E. coli} Topoisomerase III (topB) {E. coli} Topoisomerase IV subunit A (parC) {E. coli} Topoisomerase IV subunit B (parE) {E. coli} Transcription-repair coupling factor (trcF) (mfd) {E. coli} Type I restriction enzyme ecokl specific protein (hsdS) {E. coli} Type I restriction enzyme ECOR124 / 3 1M protein (hsdM) {E. coli} Type I restriction enzyme EcoRI24 / 3 1M protein (hsdM) {E. coli} Type I restriction enzyme ECOR124 / 3 R protein (hsdR) {E. coli} Type III restriction-modified ECOP15 enzyme (mod) {E. coli} Uracil DNA glycosylase (ung) {E. coli} xprB protein (xerD) {E. coli} DNA degradation Endonuclease III (nth) {E. coli} Exinuclease ABC subunit A (urvA) {E. coli} Exinuclease ABC subunit B (urvB) {E. coli} Exonuclease ABC subunit C (urvC) {E. coli} Exodeoxyribonuclease I (sboB) {E. coli} Exodeoxyribonuclease V (recB) {E. coli} Exodeoxyribonuclease V (recC) {E. coli} Exodeoxyribonuclease V (recD) {E. coli} Exonuclease III (xthA) {E. coli} Exonuclease VII, large subunit (xseA) {E. coli} Single-strand-DNA-specific exonuclease (reoJ) {E. coli} Transcription RNA synthesis, modification and DNA transcription ATP-dependent helicase HEPA (hepA) {E. coli} ATP-dependent RNA helicase (srmB) {E. coli} ATP-dependent RNA helicase DEAD (deaD) {E. coli} DNA-dependent RNA polymerase alpha chain (rpoA) {E. coli} DNA-dependent RNA polymerase beta chain (rpoB) {S. typhimurium} DNA-dependent RNA polymerase beta chain (rpoC) {E. coli} N utilization substance protein B (nusB) {E. coli} Plasmid Coby number control protein (pcnB) {E. coli} Polynucleotide phosphorylase (pnp) {E. coli} Putative ATP-dependent RNA helicase (rhlB) {E. coli} RNA polymerase omega subunit (rpoZ) {E. coli} Sigma factor (algU) {Pseudomonas aeruginosa} Transcription anti-terminator protein (nusG) {E. coli} Transcription elongation factor (greB) {E. coli} Transcription factor (nusA) {S. typhimurium} Transcription termination factor rho (rho) {E. coli} RNA degradation Anticodon nuclease blocking agent (prrD) {E. coli} Exoribonuclease II (RNasell) {E. coli} Ribonuclease D (md) {E. coli} Ribonuclease E (me) {E. Coli} Ribonuclease H (mh) {E. coli} Ribonuclease HII (EC31264) (RNASE HII) {E. coli} Ribonuclease III (rnc) {E. coli} Ribonuclease PH (rph) {E. coli} RNaseP (mpA) {E. Coli} RNaseT (mt) {E. Coli} translation Ribosomal protein synthesis and modification Ribosomal protein L1 (rpL1) {E. coli} Ribosomal protein L10 (rpL10) {S. typhimurium} Ribosomal protein L11 (rpL11) {E. coli} Ribosomal protein L11 methyltransferase (prmA) {E. coli} Ribosomal protein L13 (rpL13) {Hemophilus influenzae} Ribosomal protein L14 (rpL14) {E. coli} Ribosomal protein L15 (rpL15) {E. coli} Ribosomal protein L16 (rpL16) {E. coli} Ribosomal protein L17 (rplQ) {E. coli} Ribosomal protein L18 (rpL18) {E. coli} Ribosomal protein L19 (rpL19) {E. coli} Ribosomal protein L2 (rpL2) {E. coli} Ribosomal protein L20 (rpL20) {E. coli} Ribosomal protein L21 (rpL21) {E. coli} Ribosomal protein L22 (rpL22) {E. coli} Ribosomal protein L23 (rpL23) {E. coli} Ribosomal protein L24 (rpL24) {E. coli} Ribosomal protein L25 (rpL25) {E. coli} Ribosomal protein L27 (rpL27) {E. coli} Ribosomal protein L28 (rpL28) {E. coli} Ribosomal protein L29 (rpL29) {E. coli} Ribosomal protein L3 (rpL3) {E. coli} Ribosomal protein L30 (rpL30) {E. coli} Ribosomal protein L31 (rpL31) {E. coli} Ribosomal protein L32 (rpL32) {E. coli} Ribosomal protein L33 (rpL33) {E. coli} Ribosomal protein L34 (rpL34) {E. coli} Ribosomal protein L35 (rpL35) {E. coli} Ribosomal protein L4 (rpL4) {E. coli} Ribosomal protein L5 (rpL5) {E. coli} Ribosomal protein L6 (rpL6) {E. coli} Ribosomal protein L7 / L12 (rpL7 / L12) {E. coli} Ribosomal protein L9 (rpL9) {E. coli} Ribosomal protein S1 (rpS1) {E. coli} Ribosomal protein S10 (rpS10) {E. coli} Ribosomal protein S11 (rpS11) {E. coli} Ribosomal protein S13 (rpS13) {E. coli} Ribosomal protein S14 (rpS14) {E. coli} Ribosomal protein S15 (rpS15) {E. coli} Ribosomal protein S15 (rpS15) {E. coli} Ribosomal protein S16 (rpS16) {E. coli} Ribosomal protein S17 (rplQ) {E. coli} Ribosomal protein S18 (rpS18) {E. coli} Ribosomal protein S19 (rpS19) {E. coli} Ribosomal protein S2 (rpS2) {E. coli} Ribosomal protein S21 (rpS21) {E. coli} Ribosomal protein S3 (rpS3) {E. coli} Ribosomal protein S4 (rpS4) {E. coli} Ribosomal protein S5 (rpS5) {E. coli} Ribosomal protein S6 (rpS6) {E. coli} Ribosomal protein S6 modified protein (rimK) {E. coli} Ribosomal protein S7 (rpS7) {E. coli} Ribosomal protein S8 (rpS8) {E. coli} Ribosomal protein S9 (rpS9) {Hemophilus sonnas} Ribosome-protein-alanine acetyltransferase (riml) {E. coli} Streptomycin resistance protein (strA) {Hemophilus influenzae} Aminoacyl tRNA synthetases, tRNA modification Alanyl-tRNA synthetase (alaS) {E. coli} Arginyl-tRNA synthetase (argS) {E. coli} Asparaginyl-tRNA synthetase (asnS) {E. coli} Aspartyl-tRNA synthetase (aspS) {E. coli} cys-tRNA synthetase (cysS) {E. coli} Cysteinyl-tRNA (ser) selenium transferase (selA) {E. coli} Glutaminyl-tRNA synthetase (glnS) {E. coli} Glutamyl-tRNA synthetase (gltX) {E. coli} Glycyl-tRNA synthetase alpha chain (glyQ) {E. coli} Glycyl-tRNA synthetase beta chain (glyS) {E. coli} Histidine-tRNA synthetase (hisS) {E. coli} Isoleucyl-tRNA ligase (ileS) {E. coli} Leucyl-tKNA synthetase (leuS) {E. coli} Lysyl-tRNA synthetase (lysU) {E. coli} Lysyl-tRNA synthetase homolog (genX) {E. coli} Methionyl-tRNA formyltransferase (fmt) {E. coli} Methionyl-tRNA synthetase (metG) {E. coli} Peptidyl-tRNA hydrolase (pth) {E. coli} Phenylalanyl-tRNA synthetase beta subunit (pheS) {large Enterobacteria} Phenylalanyl-tRNA synthetase Peter subunit (pheT) {large Enterobacteria} Prolyl-tRNA synthetase (proS) {E. coli} Pseudouridylate synthetase I (hisT) {E. coli} Cuocin biosynthetic protein (queA) {E. coli} Selenium metabolic protein (selD) {E. coli} Seryl-tRNA synthetase (serS) {E. coli} Threonyl-tRNA synthetase (thrS) {E. coli} Transfer RNA-guanine transglycosylase (tgt) {E. coli} tRNA (guanine-N1) -methyltransferase (M1G-methyltrans Spherase) (trmD) {E. Coli} tRNA (uracil-5-)-methyltransferase (trmA) {E. coli} tRNA delta (2) -isoventenyl pyrophosphate transferase (trpX) {E. coli} tRNA nucleotidyltransferase (oca) {E. coli} tRNA-guanine-transglycosylase (tgt) {E. coli} Tryptophanyl-tRNA synthetase (trpS) {E. coli} Tyrosyl-tRNA synthetase (tyrS) {thiobacillus ferrooxidans} Valyl-tRNA synthetase (valS) {E. coli} Nuclear protein DNA binding protein (estimated) {Bacillus subtilis} DNA-binding protein (rdgB) {Erwinia cartobora} DNA-binding protein H-NS (hns) {E. coli} DNA-binding protein HU-ALPHA (NS2) (HU-2) {E. coli} Protein-translation and modification Disulfide oxidoreductase (por) {Hemophilus influenzae} DNA treated strand A (dprA) {E. coli} Elongation factor EF-TS (tsf) {E. coli} Elongation factor EF-Tu (duplication) (tufB) {E. coli} Elongation factor EF-Tu (duplication) (tufB) {E. coli} Elongation factor G (fusA) {E. coli} Elongation factor P (efp) {E. Coli} Glutamate-ammonia-ligase adenylyltransferase (glnE) {E. coli} Initiation factor 3 (infC) {E. coli} Initiation factor IF-1 (infA) {E. coli} Initiation factor IF-2 (infB) {E. coli} Maturation of the antibiotic MccB17 (pmbA) Methionine aminopeptidase (map) {E. coli} Oxide-reductase (dsbB) {E. coli} Peptide chain releasing factor 2 (prfB) {S. typhimurium} Peptide chain-releasing factor 3 (prfC) {E. coli} Peptidyl-prolyl cis-trans isomerase B (ppiB) {colon grip} Polypeptide chain releasing factor 1 (prfA) {murine typhimurium} Polypeptide deformylase (formylmethionine deformylase) (def) {large Enterobacteria} Ribosome releasing factor (frr) {E. coli} Rotamase, peptidylprolyl cis-trans isomerase (slyD) {large intestine Fungus} Rotamase, peptidylprolyl cis-trans isomerase (slyD) {large intestine Fungus} Transcription elongation factor (greA) {E. coli} Translation factor (selB) {E. coli} xprA protein (xprA) {E. coli} Degradation of proteins, peptides and glycopeptides Aminopeptidase A (pepA) {Richetsia plowatsekii} Aminopeptidase a / i (pepA) {E. coli} Aminopeptidase N (pepN) {E. coli} Aminopeptidase P (pepP) {E. coli} ATP-dependent protease proteolytic component (clpP) {E. coli} ATP-dependent protease ATPase subunit (clpX) {E. coli} ATP-dependent protease binding subunit (clpB) {E. coli} Collagenase activity collagenase (prtC) {porphyromonas gingivalis} HFLC protein (hflC) {E. coli} IgA1 protease (igal) {Hemophilus influenzae} IgA1 protease (igal) {Hemophilus influenzae} IgA1 protease (igal) {Hemophilus influenzae} lon protease (lon) {Bacillus brevis} Oligopeptidase A (priC) {E. coli} Peptidase D (pepD) {E. coli} Peptidase E (pepE) {E. coli} Peptidase T (pepT) {S. typhimurium} Periplasmic serine protease Do and heat shock protein (htrA) {E. coli} Putative ATP-dependent protease (sms) {E. coli} Proline dipeptidase (pepQ) {E. coli} Protease (prtH) {porphyromonas gingivalis} Protease IV (sppA) {E. coli} Phage-specific protease lambda cll inhibitor (hflK) {E. coli} Putative protease (sohB) {E. coli} Sialoglycoprotease (gcp) {Pasturella haemolytica} Transport / binding protein Amino acids, peptides, amines Arginine transport ATP-binding protein artP (artP) {E. coli} Arginine transporter permease protein (artM) {E. coli} Arginine transport system permease protein (artQ) {E. coli} Biopolymer transport protein (exbB) {Hemophilus influenzae} Biopolymer transport protein (exbD) {E. coli} Branched aa transport system II carrier protein (braB) {Pseudomonas aeruginosa} D-alanine permease (dagA) {Alteromonas haloblancchis} Dipeptide transport ATP-binding protein (dppD) {E. coli} Dipeptide transport ATP-binding protein (dppF) {E. coli} Dipeptide transport system permease protein (dppB) {E. coli} Dipeptide transport system permease protein (dppB) {E. coli} Dipeptide transport system permease protein (dppC) {E. coli} Glutamate permease (glts) {E. coli} Glutamine transporter permease protein (glnP) {E. coli} Glutamine-bound periplasmic protein (glnH) {E. coli} Leucine-specific transport protein (livG) {E. coli} Membrane-related component, LIV-II transport system (brnQ) {S. typhimurium} Glycopeptide binding protein (oppA) {E. coli} Glycopeptide binding protein (oppA) {E. coli} Glycopeptide transport ATP-binding protein (oppD) {S. typhimurium} Glycopeptide transport ATP-binding protein (oppF) {S. typhimurium} Glycopeptide transport system permease protein (oppC) C {S. typhimurium} Peptide-transporting periplasmic protein (sapA) {S. typhimurium} Peptide transport system ATP-binding protein (sapD) {S. typhimurium} Dipeptide transport system permease protein (dppC) {E. coli} Peptide transport system permease protein (sapB) {S. typhimurium} Periplasmic arginine-binding protein (artl) {Basturella haemolytica} Proton glutamate cotransport protein (gltp) {Bacillus cardotenax} Putrescine transport protein (potE) {E. coli} Serine transport factor (sdaC) {E. coli} Spermidine / putrescine transport ATP-binding protein (potA) {E. coli} Spermidine / putrescine permease protein (potB) {E. coli} Spermidine / putrescine permease protein (potC) {E. coli} Spermidine / putrescine permease protein (potD) {E. coli} Spermidine / putrescine permease protein (potD) {E. coli} Tryptophan-specific permease (mtr) {E. coli} Tyrosine-specific transport protein (tyrP) {E. coli} Tyrosine-specific transport protein (tyrP) {E. coli} Cation Pacterioferritin Komigratri-protein (bcp) {E. coli} Ferric enterobactin transporting ATP-binding protein (fepC) {E. coli} Ferric enterobactin transporting ATP-binding protein (fepC) {E. coli} Ferrichrome-iron receptor (fhuA) {E. coli} Ferritin-like protein (rsgA) {E. coli} Ferritin-like protein (rsgA) {E. coli} Iron (III) dicitrate transport ATP-binding protein FECE {E. coli} Iron (III) dicitrate transport system permease protein (fecD) {E. coli} Magnesium and pigeon transport protein (corA) {E. coli} Major ferric binding protein protein precursor (fbp) {gonococci} Mercury transport protein (merT) {Pseudomonas aeruginosa} Mercury scavenger protein (merP) {Pseudomonas fluorescens} Mercury scavenger protein (merP) {Pseudomonas fluorescens} Molybdate-bound periplasmic protein precursor (modB) {azotopacter-bi Nerangi} NA (+) / H (+) counter transport 1 (nhaA) {E. coli} NA + / H + counter-transport 1 (nhaB) {E. coli} NA + / H + Counter Transport 1 (nhaC) {Bacillus Filmas} Periplasm-binding-protein-dependent iron transport protein (sfuB) Lucessence} Periplasm-binding-protein-dependent iron transport protein (sfuC) Lucessence} Potassium efflux system (kefC) {E. coli} Potassium / copper transport ING ATPase A (copA) {Enterococcus f. Ekaris} Sodium / proline cotransport (proline permease) (putp) {E. coli} tonB protein (tonB) {Hemophilus influenzae} TRK-based potassium-absorbing protein (trkA) {E. coli} Carbohydrates, organic alcohols and acids 2-oxoglutarate / maleate transposable element (SODiT1) {Spinasia ole Lasea} D-galactose-linked periplasmic protein (mglB) {E. coli} D-xylose transport ATP-binding protein (xylG) {E. coli} D-xylose-bound periplasmic protein (rbsB) {E. coli} Enzyme 1 (PtS1) {S. typhimurium} Formic acid transport factor (formate pathway) {E. coli} Fructose-permease IIA / FPR component (fruB) {E. coli} Fructose-permease II BC component (fruA) {E. coli} Fucose operon protein (fucU) {E. coli} glpF protein (glpF) {E. coli} glpF protein (glpF) {E. coli} Gluconate permease (gntP) {Bacillus subtilis} Glucose phosphotransferase enzyme III-glc (crr) {E. coli} Glycerol-3-phosphatase transporter (glpT) {E. coli} High affinity ribose transport protein (rbsA) {E. coli} High affinity ribose transport protein (rbsC) {E. coli} High affinity ribose transport protein (rbsD) {E. coli} L-fucose permease (fucP) {E. coli} L-lactate permease (lctp) {E. coli} Lactam-utilizing protein (lamB) {Emericella nidulans} mglA protein (mglA) {E. coli} mglC protein (mglC) {E. coli} Periplasmic ribose-binding protein (rbsB) {E. coli} Phosphohistidinoprotein-hexose phosphotransferase (ptsH) {large Enterobacteria} Potassium pathway homolog (kch) {E. coli} Putative aspartate transport protein (dcuA) {E. coli} Putative aspartate transport protein (dcuA) {E. coli} Ribose transport permease protein (xylH) {E. coli} Sodium- and chloride-dependent GABA transport {Homo saviens} Sodium-dependent noradrenaline transport {Homo saviens} Nucleosides, purines and pyrimidines Ribonucleotide transporting ATP-binding protein (mkl) {Lepromycetes} Uracil permease (uraA) {E. coli} Anion Cysteine synthetase (cysZ) {E. coli} Hydrophilic membrane-bound protein (modC) {E. coli} Hydrophobic membrane-bound protein (modB) {E. coli} Integrated membrane protein (pstA) {E. coli} Nitrate transport ATPase component (nasD) {Klebsiella pneumoniae} Peripheral membrane protein B (pstB) {E. coli} Peripheral membrane protein c (pstC) {E. coli} Periplasmic phosphate-binding protein (pstS) {E. coli} Periplasmic phosphate-binding protein (pstS) {E. coli} Phosphate permease (YBR296C) {Saccharomyces cerevisiae} Other ATP-dependent transposable element homolog (msbA) {Hemophilus influenzae} ATP-binding protein (abc) {E. coli} Pustular fibrosis transmembrane conductance regulator {Boss Taurus} Heme-binding riboprotein (dppA) {Hemophilus influenzae} Heme-hemopexin-binding protein (hxA) {Hemophilus influenzae} Hemin permease (hemU) {Elnicia enterocolitica} High affinity choline transport protein (betT) {E. coli} Lactoferrin-binding protein (IbpA) {N. meningitidis} Na + / sulfate cotransport factor {Lattus norvegicus} Pantothenate permease (panF) {E. coli} Transferrin binding protein 1 precursor (tbp1) {N. meningitidis} Transferrin binding protein 1 precursor (tbp1) {N. meningitidis} Transferrin binding protein 1 precursor (tbp1) {N. meningitidis} Transferrin binding protein 2 precursor (tbp2) {N. meningitidis} Transferrin-binding protein (tfbA) {Actinobacillus pleurnew Monier} Transferrin-binding protein 1 (tbp1) {meningococcus} Transferrin-binding protein 1 (tbp2) {meningococci} Transport ATP-binding protein (cydD) {E. coli} Transport ATP-binding protein (cydD) {E. coli} Cell processing Chaperones Chaperonin (groES) (mopB) {E. coli} Heat shock protein (groEL) (mopA) {Hemophilus Juclay} Heat Striking Protein (dnaJ) {E. coli} Thermoprotein c62.5 (httpG) {E. coli} hsc66 protein (hsc66) {E. coli} hsp70 protein (dnaK) {E. coli} Cell division Cell division ATP-binding protein (ftsE) {E. coli} Cytostatic factor (sulA) {Vibrio chorele} Cell division protein (ftsA) {E. coli} Cell division protein (ftsH) {E. coli} Cell division protein (ftsH) {E. coli} Cell division protein (ftsJ) {E. coli} Cell division protein (ftsL) {E. coli} Cell division protein (ftsQ) {E. coli} Cell division protein (ftsW) {E. coli} Cell division protein (ftsY) {E. coli} Cell division protein (ftsZ) {E. coli} Cell division protein (mukB) {E. coli} Cytoplasmic filament protein (cafA) {E. coli} ftsX protein (ftsX) {E. coli} mukB inhibitor protein (smbA) {E. coli} Penicillin-binding protein 3 (ftsl) {E. coli} Protein and peptide secretion GTP-binding membrane protein (lepA) {E. coli} Colicin V secreted ATP-binding protein (cvaB) {E. coli} Riboprotein signal peptidase (lspA) {E. coli} Peptide transport system ATP-binding protein SAPF (sapF) {E. coli} Preprotein translocase (secE) {E. coli} Preprotein translocase SECY subunit (secY) {E. coli} Protein-transport membrane protein (secD) {E. coli} Protein-transport membrane protein (secF) {E. coli} Protein-transport membrane protein (secG) {E. coli} Protein-transport protein (secB) {E. coli} secA protein (secA) {E. coli} Signal peptidase I (lepB) {E. coli} Signal recognition particle protein (54 homologues) (ffh) {E. coli} Initiation factor (tig) {E. coli} Type 4 prepirin-like protein-specific leader peptidase (hopD) {E. coli} xopS protein (xcpS) {Pseudomonas buchida} Detoxification KW20 catalase (hktE) {Hemophilus influenzae} Superoxide dismutase (sodA) {Hemophilus influenzae} Thiophene and furan oxidized protein (thdF) {E. coli} Cell death Hemolysin (tlyc) {Serprina hyodysenteriae} Hemolysin, 21kDa (hly) {Actinobacillus prouroneumoniae} Dead protein (kicA) {E. coli} Killed protein inhibitor (kicB) {E. coli} Leukotoxin secreting ATP-binding protein (lktB) {Actinobacillus acti Nomiseten Committance} Conversion com101A protein (comF) {Hemophilus influenzae} Reaction locus E (comE1) {Bacillus subtilis} tfoX protein (tfoX) {Hemophilus influenzae} Converted gene group hypothetical protein (GB: M62809_1) (com) {Hemophilus i Influenza} Converted gene group hypothetical protein (GB: M62809_10) (com) {Hemophilus Influenza} Converted gene group hypothetical protein (GB: M62809_2) (com) {Hemophilus i Influenza} Converted gene group hypothetical protein (GB: M62809_3) (com) {Hemophilus i Influenza} Converted gene group hypothetical protein (GB: M62809_4) (com) {Hemophilus i Influenza} Converted gene group putative protein (GB: M62809_5) (com) {Hemophilus i Influenza} Transformed gene group hypothetical protein (GB: M62809_6) (com) {Hemophilus i Influenza} Converted gene group hypothetical protein (GB: M62809_7) (com) {Hemophilus i Influenza} Other classes Colicin-related functions Colicin resistance protein (tolB) {E. coli} Colicin V-producing protein (pur regulon) (cvpA) {E. coli} Inner membrane protein (tolQ) {E. coli} Inner membrane protein (tolR) {E. coli} Outer membrane integration protein (tolA) {E. coli} Outer membrane integration protein (tolA) {E. coli} Phage-related functions and bophages E16 protein (muE16) {bacteriophage mu} G protein (muG) {bacteriophage mu} G protein (muG) {bacteriophage mu} gam protein {bacteriophage mu} Heat shock protein B253 (grpE) {E. coli} Host factor-1 (HF-1) (hfq) {E. coli} I protein (mul) {bacteriophage mu} MuB protein (muB) {bacteriophage mu} N protein (muN) {bacteriophage mu} P protein {bacteriophage mu} Terminase subunit 1 {Bacteriophage SF6} Transposase A (muA) {Bacteriophage mu} Transposons-related functions Insertion sequence IS1016 (V-4) hypothetical protein (GB: X58176_2) {hemophyte Russ Influenza} IS1016-V6 protein (IS1016-V6) {Hemophilus influen Ze} IS1016-V6 protein (IS1016-V6) {Hemophilus influen Ze} IS1016-V6 protein (IS1016-V6) {Hemophilus influen Ze} Drug / homolog susceptibility Acriflavine resistance protein (acrB) {E. coli} Protein for ampD signal (ampD) {E. coli} Bicyclomycin resistance protein (bor) {E. coli} Mercury resistance regulatory protein (merR2) {thiobacillus ferrooxidans} Regulator of drug activity (mda66) {E. coli} Complex drug resistance protein (emrB) {E. coli} Complex drug resistance protein (emrA) {E. coli} Complex drug resistance protein (mdl) {E. coli} Nodule protein T (nodT) {Rhizobium reguminosarum} rRNA (adenosine-N6, N6-)-dimethyltransferase (ksgA) { E. coli} Telluric acid resistant protein (tehA) {E. coli} Telluric acid resistant protein (tehB) {E. coli} Radiation sensitivity radC protein (radC) {E. coli} Adaptive, atypical conditions In-growth protein (auto) {Alkalines eutrophus} Heat shock protein (httpX) {E. coli} Heat shock protein B (ibpB) {E. coli} htrA-like protein (htrH) {E. coli} Invasion protein (invA) {Bartonella basiliformis} NAD (P) H: menadione oxidoreductase {mus musculus} Survival protein (surA) {E. coli} uspA protein (uspA) {E. coli} Virulence plasmid protein (vagC) {Salmonella Dublin} Virulence-related protein A (vapA) {Dikeobacter nodosas} Virulence-related protein C (vapC) {Dikeobacter nodosas} Virulence-related protein C (vapC) {Dikeobacter nodosas} Virulence-related protein D (vapD) {Dikeobacter nodosas} Virulence plasmid protein (mlgA) {Shewanella colwariana} Unidentified 15kDa protein (P15) {E. coli} 2-hydroxy acid dehydrogenase homolog (ddh) {Dimomonas mobilis} Beta-lactamase regulatory homolog (mazG) {E. coli} Conjugation co-suppressor (finO) {E. coli} Delta-1-pyrroline-5-carboxylate reductase (proC) {Pseudomonas aeruginosa} devA protein (devA) {Anabaena species} devB protein (devB) {Anabena species} Embryo wealthy protein, group 3 {Triticum estivum} Extragenic repressor (suhB) {E. coli} GCPE protein (protein E) (gpcE) {E. coli} GerC2 protein (gerC2) {Bacillus subtilis} glpX protein (glpX) {E. coli} Glyoxylic acid-derived protein {E. coli} hslU protein (hslU) {E. coli} hslV protein (hslV) {E. coli} ilv-related protein {E. coli} Isochorismate synthase (entC) {Bacillus subtilis} Membrane-related ATRase (cbiO) {Propionibacterium leudenleyii} Membrane protein (lapB) {Basteurella haemolytica} Membrane protein (lapB) {Basteurella haemolytica} N-carbamyl-L-amino acid amide hydrolase {Bacillus stearothermo Philas} Nitrogen fixing protein (nifS) {Anabaena species} Nitrogen-fixing protein (nifS) {Mycobacterium leprea} Nitrogen-fixing protein (nifS) {Mycobacterium leprea} Nitrogen fixing protein (nifU) {Klebsiella pneumoniae} Nitrogen fixation protein (nnfE) {Lordobacter capsalatas} Nitrogen fixation protein (nnfE) {Lordobacter capsalatas} Nitrogenase C (nifC) {Clostilidium pasteurianum} Nitrogenase C (nifC) {Clostilidium pasteurianum} nmt1 protein (nmt1) {Aspergillus pasteurianum} Partitioning system protein (parB) {plasmid RP4} larD protein (larD) {E. coli} larD protein (larD) {E. coli} skp protein (skp) {pasteurella maltosida} Small protein (smpB) {E. coli} spollE protein (spillE) {Coxiella pumeti} Suppressor protein (msgA) {E. coli} Surfactin (sfpo) {Bacillus subtilis} toxR legron (tagD) {Vibrio chorele} traN protein (traN) {plasmid RP4} Transport ATP-binding protein (cydC) {E. coli} Transport ATP-binding protein (cydC) {E. coli} vanH protein (vanH) {transposon Tn1546} Mucus state locus protein (mucB) {Pseudomonas aeruginosa} Phenol hydroxylase (ORF6) {Acinetobacter calcoaceticus} Plasma protease C1 inhibitor {Homo sapiens} Known ATP-dependent rearrangement homolog (msbA) Outer membrane protein P2 (ompP2) Single-stranded DNA binding protein (ssb) tonB protein (tonB) Heme-hemopexin-binding protein (hxuA) Adenylate kinase (ATP-AMP transphosphorylase) (adk) Hypothetical protein (SP: P24326) udp-glucose 4-epimerase (galactowardenase) (galE) Hypothetical protein (SP: P24324) PC protein (15 kd peptidoglycan-related outer membrane riboprotein) (pal) Outer membrane protein P1 (ompP1) Converted gene group hypothetical protein (GB: M62809_7) (com) Converted gene group hypothetical protein (GB: M62809_6) (com) Converted gene group hypothetical protein (GB: M62809_5) (com) Converted gene group hypothetical protein (GB: M62809_4) (com) Converted gene group hypothetical protein (GB: M62809_3) (com) Converted gene group hypothetical protein (GB: M62809_2) (com) Converted gene group hypothetical protein (GB: M62809_1) (com) Hinoll endonuclease (Hincll) Modified methylase Hincl (hinclM)   Lipooligosaccharide biosynthetic protein   Streptomycin resistance protein (strA)   Recombinase (recA) tfoX protein (tfoX) Adenylate cyclase (cyaA) 28 kDa membrane protein (hlpA) Protein D (hpd) Riboprotein (hel) Aldose 1-epimerase precursor (mutalotase) (mro) Galactokinase (galK) Galactose-1-phosphate uridylyltransferase (galT) Galactose operon inhibitor (galS) Hypothetical protein (GB: M94205_1) Disulfide oxidoreductase (por) Heme-binding riboprotein (dppA) Protective surface antigen D15 KW20 catalase (hktE) Cyclic AMP receptor protein (crp) Superoxide dismutase (sodA) Outer membrane protein P5 (ompA) DNA helicase II (uvrD) Hindll modified methyltransferase (hindllM) Hindll restriction endonuclease (hindllR) DNA polymerase III, chi subunit (holC) lic-1 operon protein (licC) lic-1 operon protein (licD) 15 kd peptidoglycan-related riboprotein (1 pp) Formyltetrahydrofolate hydrolase (purU) Enol pyruvyl shikimate phosphate synthase (aroA) lsg locus hypothetical protein (GB: M94855_8) lsg locus hypothetical protein (GB: M94855_7) Iss gene locus hypothetical protein (GB: M94855_6) Iss gene locus hypothetical protein (GB: M94855_5) lsg locus hypothetical protein (GB: M94855_4) lsg locus hypothetical protein (GB: M94855_3) lsg locus hypothetical protein (GB: M94855_2) lsg locus hypothetical protein (GB: M94855_1)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) C07K 16/08 (31) Priority claim number 08 / 487,429 (32) Priority date June 7, 1995 (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG) , UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, E, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventor Freischmann, Robert Di Adams, Mark Di, United States 20878 Maryland, Gaithersburg, Gaithersburg, United States No. 470 (72) Inventor Daffef Drive, No. 15205 (72) Inventor White, Owen United States 20878 Queens Orchard Boulevard, Gaithersburg, MD Apartment No. 886 Apartment Number 202 (72) Inventor Smith, Ha Milton-Oh, U.S.A., Carbridge Circle 8222, Towson, Maryland 21204, United States Inventor Vent, Jay Craig United States 20854 Glen Mill Road, Potomac, Maryland 11985

Claims (1)

[Claims]   1. SEQ ID NO: 1. Nucleotide sequence represented by 1, a representative fragment thereof Or at least 99.9% identical to the nucleotide sequence represented by SEQ ID NO: 1. A computer-readable medium recording a nucleotide sequence.   2. Except for the fragment of SEQ ID NO: 1 shown in Table 1b, No. 1 of the fragment of SEQ ID NO: 1 or a degenerate variant thereof is described. A computer-readable medium recorded.   3. The above media is floppy disk, hard disk, random access memo Group consisting of memory (ROM), read-only memory (ROM) and CD-ROM The computer-readable medium according to claim 1, wherein the medium is selected from the group consisting of:   4. The above media is floppy disk, hard disk, random access memo Group consisting of memory (ROM), read-only memory (ROM) and CD-ROM The computer-readable medium according to claim 3, which is selected from the group consisting of:   5. The following elements:   a) The nucleotide sequence of SEQ ID NO: 1, a representative fragment or sequence thereof Column identification number: a nucleotide sequence at least 99.9% identical to the nucleotide sequence 1 Data storage means comprising:   b) In order to identify the homologous sequence (s), the target sequence is Search means for comparing with the nucleotide sequence of data storage means, and   c) Extractor for obtaining the homologous sequence (s) of step (b) above Dan, To identify commercially important fragments of the Haemophilus genome containing Computer-based system.   6. SEQ ID NO: 1 is a nucleotide sequence represented by At least 99.9% identical to the nucleotide sequence of fragment or SEQ ID NO: 1 A database containing the nucleotide sequence of Hemophile comprising obtaining a nucleic acid molecule comprising a nucleotide sequence complementary to the sequence A method of identifying a commercially important nucleic acid fragment of the R. genus, comprising the steps of: Sequences are not randomly selected.   7. SEQ ID NO: 1. Nucleotide sequence represented by 1, a representative fragment thereof Nucleotide at least 99.9% identical to the nucleotide sequence of SEQ ID NO: 1 The database containing the peptide sequence is compared with the target sequence and complemented with the target sequence. Of obtaining a nucleic acid molecule comprising a specific nucleotide sequence A method for identifying an expression control fragment of a gene, wherein the target sequence regulates gene expression. Contains sequences known to knot.   8. Isolated protein-encoding nucleic acid fragment of the Rd genome of Haemophilus influenzae Wherein the fragment is the fragment of SEQ ID NO: 1 shown in Table 1b. Except for any one of the fragments of SEQ ID NO: 1 shown in Table 1a It consists of a nucleotide sequence or a degenerate variant thereof.   9. Except for the fragment of SEQ ID NO: 1 shown in Table 1b, Any one of the fragments of the Rd genome or the degeneracy thereof A vector containing the variant.   10. An isolated fragment of the Rd genome of Haemophilus influenzae, wherein the fragment The fragment modulates the expression of an operably linked open reading frame and the fragment Is shown in Table 1a except for the fragment of SEQ ID NO: 1 shown in Table 1b. Length of about 10 to 200 bases in the 5 'of any one of the open reading frames It consists of a nucleotide sequence or a degenerate variant thereof.   11. 9. Any of the fragments of the Rd genome of Haemophilus influenzae according to claim 8. Or a vector containing one.   12. 9. Any one of the fragments of the genome of the genus Haemophilus according to claim 8. An organism that has been modified to contain   13. Any one of the fragments of the genome of the genus Haemophilus according to claim 10. An organism that has been modified to contain one.   14． 5 'of a nucleic acid molecule consisting of a nucleotide sequence of about 10 to 100 bases , Except for the fragment of SEQ ID NO: 1 shown in Table 1b. 5 of any one of the fragments of the genome of the genus Haemophilus or a degenerate variant thereof. '. The method for regulating the expression of the above nucleic acid, comprising the step of binding to   15. Except for the fragment of SEQ ID NO: 1 shown in Table 1b, Encodes a homologue of any one of the indicated fragments of the Haemophilus genome An isolated nucleic acid molecule comprising:   a) replacing any one of the fragments of the Haemophilus genome shown in Table 1a Screening a genomic DNA library to use as a target sequence;   b) The library containing a sequence that hybridizes with the target sequence Identifying members of   c) isolating the nucleic acid molecule from the member identified in step (b); Manufactured by stages.   16. Except for the fragment of SEQ ID NO: 1 shown in Table 1b, Encodes a homologue of any one of the indicated fragments of the Haemophilus genome An isolated DNA molecule, wherein the nucleic acid molecule is:   a) isolating mRNA, DNA or cDNA produced from the organism;   b) the nucleotide sequence of the nucleic acid molecule is the flag of the Haemophilus genome Amplification primers that are derived from the primer and prime the amplification Amplify nucleic acid molecules homologous to   c) isolating the sequence produced and amplified in step (b). Manufactured by stages.   17． Except for the fragment of SEQ ID NO: 1 shown in Table 1b, Any one of the indicated fragments of the Rd genome or the fragment An isolated polypeptide encoded by a degenerate variant of a fragment.   18. An isolated polynucleotide encoding any one of the polypeptides of claim 17. Nucleotide molecule.   19. An antibody that selectively binds to any one of the polypeptides of claim 17.   20. a) The nucleotide sequence of the heterologous nucleic acid molecule is the sequence identification number shown in Table 1b. No. 1 except for the fragment of Rd genome of Haemophilus influenzae shown in Table 1a. A heterologous nucleic acid molecule comprising any one of the fragments or a degenerate variant thereof. The containing host is subjected to conditions for expressing the heterologous nucleic acid molecule to produce a protein. Incubate with;   b) isolating the protein; A method of producing a polypeptide in a host cell comprising a step.