Free online classes johns hopkins,online course free certificate,high explosives will create what at the point of origin - Downloads 2016

Nucleotide sequence of the Haemophilus influenzae Rd genome, fragments thereof, and uses thereof - Human Genome Sciences, Inc.
The present invention provides the sequencing of the entire genome of Haemophilus influenzae Rd, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use.
An isolated polynucleotide comprising the nucleic acid sequence of ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1.
The isolated polynucleotide of claim 1, wherein said polynucleotide comprises a heterologous polynucleotide sequence. The isolated polynucleotide of claim 2, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.
A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 1 into a vector. The recombinant vector of claim 6, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
The recombinant host cell of claim 8, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression. An isolated polynucleotide for the detection of Haemophilus influenzae, wherein said isolated polynucleotide comprises at least 18 contiguous nucleotides of the nucleic acid sequence of ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1. The isolated polynucleotide of claim 10, wherein said polynucleotide comprises a heterologous polynucleotide sequence. The isolated polynucleotide of claim 11, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 10 into a vector. The recombinant vector of claim 15, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
The recombinant host cell of claim 17, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression. The isolated polynucleotide of claim 10, wherein said isolated polynucleotide comprises at least 20 contiguous nucleotides of the nucleic acid sequence of ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1. The isolated polynucleotide of claim 10, wherein said isolated polynucleotide comprises at least 40 contiguous nucleotides of the nucleic acid sequence of ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1.
In addition to the entire genomic sequence, the present invention identifies over 1700 protein encoding fragments of the genome and identifies, by position relative to a unique Not I restriction endonuclease site, any regulatory elements which modulate the expression of the protein encoding fragments of the Haemophilus genome. The present invention discloses compositions comprising the nucleotide sequence of Haemophilus influenzae, fragments thereof and usage in industrial fermentation and pharmaceutical development.BACKGROUND OF THE INVENTIONThe complete genome sequence from a free living cellular organism has never been determined. No one has attempted to determine sequences of the order of a megabase or more by a random shotgun approach.H. It is a resident of the upper respiratory mucosa of children and adults and causes otitis media and respiratory tract infections mostly in children. The most serious complication is meningitis, which produces neurological sequelae in up to 50% of affected children. Serotype b accounts for the majority of human disease.Interest in the medically important aspects of H. A number of the genes responsible for the capsular polysaccharide have been mapped and sequenced (Kroll et al., Mol. Several outer membrane protein (OMP) genes have been identified and sequenced (Langford et al., J.
The lipoligosaccharide (LOS) component of the outer membrane and the genes of its synthetic pathway are under intensive study (Weiser et al., J. While a vaccine has been available since 1984, the study of outer membrane components is motivated to some extent by the need for improved vaccines. Recently, the catalase gene was characterized and sequenced as a possible virulence-related gene-(Bishni et al., in press). USA 89:1626-1630 (1992)), at least two are involved in recombination processes (Barouki and Smith, J. USA 89:10252-10256 (1992)), where they appear to function as structural components or in the assembly of the DNA transport machinery. Various genes have-been mapped to restriction fragments by Southern hybridization probing of restriction digest DNA bands.
This map will be valuable in verification of the assembly of a complete genome sequence from randomly sequenced fragments. About half are from serotype b and half from Rd.SUMMARY OF THE INVENTIONThe present invention is based on the sequencing of the Haemophilus influenzae Rd genome.
In one application of this embodiment, the sequences of the present invention are recorded on computer readable media. Such systems are designed to identify commercially important fragments of the Haemophilus influenzae Rd genome.Another embodiment of the present invention is directed to isolated fragments of the Haemophilus influenzae Rd genome.
The fragments of the Haemophilus influenzae Rd genome of the present invention include, but are not limited to, fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in a sample, hereinafter, diagnostic fragments (DFs).Each of the ORF fragments of the Haemophilus influenzae Rd genome disclosed in Tables 1(a) and 2, and the EMF found 5? to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers for the presence of a specific microbe in a sample, for the production of commercially important pharmaceutical agents, and to selectively control gene expression.The present invention further includes recombinant constructs comprising one or more fragments of the Haemophilus influenzae Rd genome of the present invention. The recombinant constructs of the present invention comprise vectors, such as a plasmid or viral vector, into which a fragment of the Haemophilus influenzae Rd has been inserted.The present invention further provides host cells containing any one of the isolated fragments of the Haemophilus influenzae Rd genome of the present invention. The host cells can be a higher eukaryotic host such as a mammalian cell, a lower eukaryotic cell such as a yeast cell, or can be a procaryotic cell such as a bacterial cell.The present invention is further directed to isolated proteins encoded by the ORFs of the present invention. A variety of methodologies known in the art can be utilized to obtain any one of the proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. In an alternative method, the protein is purified from bacterial cells which naturally produce the protein.
Lastly, the proteins of the present invention can alternatively be purified from cells which have been altered to express the desired protein.The invention further provides methods of obtaining homologs of the fragments of the Haemophilus influenzae Rd genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention. Such antibodies include both monoclonal and polyclonal antibodies.The invention further provides hybridomas which produce the above-described antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.The present invention further provides methods of identifying test samples derived from cells which express one of the ORF of the present invention, or homolog thereof. Specifically, such agents include antibodies (described above), peptides, carbohydrates, pharmaceutical agents and the like. Many fragments of the Haemophilus influenzae Rd genome will be immediately identified by similarity searches against GenBank or protein databases and will be of immediate value to Haemophilus researchers and for immediate commercial value for the production of proteins or to control gene expression. The PHA synthase that synthesizes this polymer has been identified and sequenced in a number of bacteria, none of which are evolutionarily close to H. However, the genomic sequence of the present invention allows the identification of the gene by utilizing search means described below.Developing the methodology and technology for elucidating the entire genomic sequence of bacterial and other small genomes has and will greatly enhance the ability to analyze and understand chromosomal organization. In particular, sequenced genomes will provide the models for developing tools for the analysis of chromosome structure and function, including the ability to identify genes within large segments of genomic DNA, the structure, position, and spacing of regulatory elements, the identification of genes with potential industrial applications, and the ability to do comparative genomic and molecular phylogeny.DESCRIPTION OF THE FIGURESFIG. 2—Block diagram of a computer system 102 that can be used to implement the computer-based systems of present invention.FIG. Factura (AB) is a Macintosh program designed for automatic vector sequence removal and end trimming of sequence files. The program esp runs on a Macintosh platform and parses the feature data extracted from the sequence files by Factura to the Unix based H. Assembly is accomplished by retrieving a specific set of sequence files and their associated features using stp, an X-windows graphical interface and control program which can retrieve sequences from the H. The sequence files were assembled using TIGR Assembler, an assembly engine designed at TIGR for rapid and accurate assembly of thousands of sequence fragments. TIGR Editor is a graphical interface which can parse the aligned sequence files from TIGR Assembler output and display the alignment and associated electropherograms for contig editing. Identification of putative coding regions was performed with Genemark (Borodovsky and McIninch, Computers Chem. 17(2):123 (1993)), a Markov and Bayes modeled program for predicting gene locations, and trained on a H. Peptide searches were performed against the three reading frames of each Genemark predicted coding region using blaze (Brutlag et al., Computers Chem.
Optimal protein alignments were obtained using the program praze which extends alignments across potential frameshifts.
The output was inspected using a custom graphic viewing program, gbyob, that interacts directly with the H. The alignments were further used to identify potential frameshift errors and were targeted for additional editing.FIG. Outer concentric circle: The location of each identified coding region for which a gene identification was made.
Over 300 lambda clones were sequenced from each end to confirm the overall structure of the genome and identify the 6 ribosomal operons. Fourth concentric circle: The locations of the 6 ribosomal operons, the tRNAs and the cryptic mu-like prophage. The locations of the following repeats are shown: CTGGCT, GTCT, ATT, AATGGC, TFGA, TTGG, TITA, TTATC, TGAC, TCGTC, AACC, TTGC, CAAT, CCAA. The putative origin of replication is illustrated by the outward pointing arrows originating near base 603,000. Two potential termination sequences are shown near the opposite midpoint of the circle.FIGS. However in the non-infectious Rd strain the 8 genes of the fimbrial gene cluster have been excised.


A 172 bp spacer region is located in this region in the Rd strain and continues to be flanked by the pepN and purE genes.FIG. The amino acid sequences of five predicted coding regions that do not display homology with known peptide sequences (GenBank release 87), each exhibit multiple hydrophobic domains that are characteristic of channel-forming proteins.
The predicted coding region sequences were analyzed by the Kyte-Doolittle algorithm (Kyte and Doolittle, J. Preferred representative fragments of the present invention are Haemophilus influenzae open reading frames, expression modulating fragments, uptake modulating fragments, and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in sample. In very rare instances, this may reveal a nucleotide sequence error present in the nucleotide sequence disclosed in SEQ ID NO:1.
As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.
A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.As used herein, “recorded” refers to a process for storing information on computer readable medium.
A skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.
The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. 17:203-207(1993)) search algorithms on a Sybase system was used to identify open reading frames (ORFs) within the Haemophilus influenzae Rd genome which contain homology to ORFs or proteins from other organisms. Such ORFs are protein encoding fragments within the Haemophilus influenzae Rd genome and are useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. Such systems are designed to identify commercially important fragments of the Haemophilus influenzae Rd genome.As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention.As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
Search means are used to identify fragments or regions of the Haemophilus influenzae Rd genome which match a particular target sequence or target motif.
A variety of known algorithms are disclosed publicly and a variety of commercially available-software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids.
A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.
The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that searches for commercially important fragments of the Haemophilus influenzae Rd genome, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. Protein target motifs include, but are not limited to, enzymic active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
A preferred format for an output means ranks fragments of the Haemophilus influenzae Rd genome possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of the Haemophilus influenzae Rd genome.
In the present examples, implementing software which implement the BLAST and BLAZE algorithms (Altschul et al., J.
215:403-410 (1990)) was used to identify open reading frames within the Haemophilus influenzae Rd genome. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer-based systems of the present invention.One application of this embodiment is provided in FIG. 2 provides a block diagram of a computer system 102 that can be used to implement the present invention. Also connected to the bus 104 are a main memory 108 (preferably implemented as random access memory, RAM) and a variety of secondary storage devices 110, such as a hard drive 112 and a removable medium storage device 114.
The removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc.
The fragments of the Haemophilus influenzae Rd genome of the present invention include, but are not limited to fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in a sample, hereinafter diagnostic fragments (DFs).As used herein, an “isolated nucleic acid molecule” or an “isolated fragment of the Haemophilus influenzae Rd genome” refers to a nucleic acid molecule possessing a specific nucleotide sequence which has been subjected to purification means to reduce, from the composition, the number of compounds which are normally associated with the composition. A variety of purification means can be used to generated the isolated fragments of the present invention. These include, but are not limited to methods which separate constituents of a solution based on charge, solubility, or size.In one embodiment, Haemophilus influenaze Rd DNA can be mechanically sheared to produce fragments of 15-20 kb in length. These fragments can then be used to generate an Haemophilus influenzae Rd library by inserting them into labda clones as described in the Examples below. Primers flanking, for example, an ORF provided in Table 1(a) can then be generated using nucleotide sequence information provided in SEQ ID NO:1. In particular, Table 1a indicates the location of ORFs within the Haemophilus influenzae genome which encode the recited protein based on homology matching with protein sequences from the organism appearing in parentheticals (see the fourth column of Table 1(a)).The first column of Table 1(a) provides the “GeneID” of a particular ORF. One of ordinary skill will recognize that ORFs may be oriented in opposite directions in the Haemophilus influenae genome.
The concepts of percent identity and percent similarity of two polypeptide sequences is well understood in the art. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments which induce the expression or an operably linked ORF in response to a specific regulatory factor or physiological event.
An intergenic segment, or a fragment of the intergenic segment, from about 10 to 200 nucleotides in length, taken 5? from any one of the ORFs of Tables 1(a), 1(b), or 2 will modulate the expression of an operably linked 3? ORF in a fashion similar to that found with the naturally linked ORF sequence.
As used herein, an “intergenic segment” refers to the fragments of the Haemophilus genome which are between two ORF(s) herein described.
Alternatively, EMFs can be identified using known EMFs as a target sequence or target motif in the computer-based systems of the present invention.The presence and activity of an EMF can be confirmed using an EMF trap vector.
A marker sequence encodes an identifiable phenotype, such as antibiotic resistance or a complementing nutrition auxotrophic factor, which can be identified or assayed when the EMF trap vector is placed within an appropriate host under appropriate conditions. As described above, a EMF will modulate the expression of an operably linked marker sequence.
A more detailed discussion of various marker sequences is provided below.A sequence which is suspected as being a EMF is cloned in all three reading frames in one or more restriction sites upstream from the marker sequence in the EMF trap vector. The vector is then transformed into an appropriate host using known procedures and the phenotype of the transformed host in examined under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence.As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotide molecules which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described above.The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.
The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence. DFs can be readily identified by identifying unique sequences within the Haemophilus influenzae Rd genome, or by generating and testing probes or amplification primers consisting of the DF sequence in an appropriate diagnostic format which determines amplification or hybridization selectivity.The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic and species variations thereof. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.
Alternatively, error screening can be performed by sequencing corresponding polynucleotides of Haemophilus influenzae origin isolated by using part or all of the fragments in question as a probe or primer.Each of the ORFs of the Haemophilus influenzae Rd genome disclosed in Tables 1(a), 1(b) and 2, and the EMF found 5? to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers to detect the presence of a specific microbe, such as Haemophilus influenzae RD, in a sample. This is especially the case with the fragments or ORFs of Table 2, which will be highly selective for Haemophilus influenzae.
In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.
Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.The present invention further provides recombinant constructs comprising one or more fragments of the Haemophilus influenzae Rd genome of the present invention.
The recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a fragment of the Haemophilus influenzae Rd has been inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
For vectors comprising the EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF or UMF.


Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the artsThe present invention further provides host cells containing any one of the isolated fragments of the Haemophilus influenzae Rd genome of the present invention, wherein the fragment has been introduced into the host cell using known transformulation methods. The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a procaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L.
Preferred nucleic acid fragments of the present invention are the ORFs depicted in Table 1(a) which encode proteins.A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. This is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polpeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.
These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.
These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Generally, DNA segments encoding the polypeptides and proteins provided by this invention are assembled from fragments of the Haemophilus influenzae Rd genome and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.“Recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. The expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.“Recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extra chromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis., USA).
These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.Following transformation of a suitable host strain and growth of the host. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5? flanking nontranscribed sequences.
DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.Recombinant polypeptides and proteins produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.The present invention further includes isolated polypeptides, proteins and nucleic acid molecules which are substantially equivalent to those herein described. As used herein, substantially equivalent can refer both to nucleic acid and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, sequences having equivalent biological activity, and equivalent expression characteristics are considered substantially equivalent. For purposes of determining equivalence, truncation of the mature sequence should be disregarded.The invention further provides methods of obtaining homologs from other strains of Haemophilus influenzae, of the fragments of the Haemophilus influenzae Rd genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention.
As used herein, a sequence or protein of Haemophilus influenzae is defined as a homolog of a fragment of the Haemophilus influenzae Rd genome or a protein encoded by one of the ORFs of the present invention, if it shares significant homology to one of the fragments of the Haemophilus influenzae Rd genome of the present invention or a protein encoded by one of the ORFs of the present invention. Tables 1(a) further provides an identification of the type of polypeptide which is encoded for by each ORF. Biosynthetic EnzymesOpen reading frames encoding proteins involved in mediating the catalytic reactions involved in intermediary and macromolecular metabolism, the biosynthesis of small molecules, cellular processes and other functions includes enzymes involved in the degradation of the intermediary products of metabolism, enzymes involved in central intermediary metabolism, enzymes involved in respiration, both aerobic and anaerobic, enzymes involved in fermentation, enzymes involved in ATP proton motor force conversion, enzymes involved in broad regulatory function, enzymes involved in amino acid synthesis, enzymes involved in nucleotide synthesis, enzymes involved in cofactor and vitamin synthesis, can be used for industrial biosynthesis. The various metabolic pathways present in Haemophilus can be identified based on absolute nutritional requirements as well as by examining the various enzymes identified in Table 1(a).Identified within the category of intermediary metabolism, a number of the proteins encoded by the identified ORFs in Tables 1(a) are particularly involved in the degradation of intermediary metabolites as well as non-macromolecular metabolism. Some of the enzymes identified include amylases, glucose oxidases, and catalase.Proteolytic enzymes are another class of commercially important enzymes. Proteolytic enzymes find use in a number of industrial processes including the processing of flax and other vegetable fibers, in the extraction, clarification and depectinization of fruit juices, in the extraction of vegetables' oil and in the maceration of fruits and vegetables to give unicellular fruits.
Enzymes involved in the degradation of these sugars can be used in industrial fermentation. Some of the important sugar transforming enzymes, from a commercial viewpoint, include sugar isomerases such as glucose isomerase. Other metabolic enzymes have found commercial use such as glucose oxidases which produces ketogulonic acid (KGA).
KGA is an intermediate in the commercial production of ascorbic acid using the Reichstein's procedure (see Krueger et al., Biotechnology 6(A), Rhine, H. The most important application of GOD is the industrial scale fermentation of gluconic acid. Market for gluconic acids which are used in the detergent, textile, leather, photographic, pharmaceutical, food, feed and concrete industry (see Bigelis in Gene Manipuilions and Fungi, Benett, J. In addition to industrial applications, GOD has found applications in medicine for quantitative determination of glucose in body fluids recently in biotechnology for analyzing syrups from starch and cellulose hydrosylates. In the field of industrial enzymes, the glucose isomerase process shows the largest expansion in the market today. Initially, soluble enzymes were used and later immobilized enzymes were developed (Krueger et al., Biotechnology, The Textbook of Industrial Microbiology, Sinauer Associated Incorporated, Sunderland, Mass. Today, the use of glucose-produced high fructose syrups is by far the largest industrial business using immobilized enzymes.
Because of their industrial importance, there is a large body of published and unpublished information regarding the use of these enzymes in industrial processes. A major use of lipases is in the fat and oil industry for the production of neutral glycerides using lipase catalyzed inter-esterification of readily available triglycerides. Application of lipases include the use as a detergent additive to facilitate the removal of fats from fabrics in the course of the washing procedures.The use of enzymes, and in particular microbial enzymes, as catalyst for key steps in the synthesis of complex organic molecules is gaining popularity at a great rate.
Preparation of chiral intermediates is of interest to a wide range of synthetic chemists particularly those scientists involved with the preparation of new pharmaceuticals, agrochemicals, fragrances and flavors.
The following reactions catalyzed by enzymes are of interest to organic chemists: hydrolysis of carboxylic acid esters, phosphate esters, amides and nitriles, esterification reactions, trans-esterification reactions, synthesis of amides, reduction of alkanones and oxoalkanates, oxidation of alcohols to carbonyl compounds, oxidation of sulfides to sulfoxides, and carbon bond forming reactions such as the aldol reaction.
When considering the use of an enzyme encoded by one of the ORFs of the present invention for biotransformation and organic synthesis it is sometimes necessary to consider the respective advantages and disadvantages of using a microorganism as opposed to an isolated enzyme.
Pros and cons of using a whole cell system on the one hand or an isolated partially purified enzyme on the other hand, has been described in detail by Bud et al., Chemistry in Britain (1987), p. 127.Amino transferases, enzymes involved in the biosynthesis and metabolism of amino acids, are useful in the catalytic production of amino acids. The advantages of using microbial based enzyme systems is that the amino transferase enzymes catalyze the stereo-selective synthesis of only l-amino acids and generally possess uniformly high catalytic rates.
A variety of commercially important enzymes have previously been isolated from members of Haemophilus sp.
Table 1(a) identifies a wide array of enzymes, such as restriction enzymes, ligases, gyrases and methylases, which have immediate use in the biotechnology industry.2. Generation of AntibodiesAs described here, the proteins of the present invention, as well as homologs thereof, can be used in a variety procedures and methods known in the art which are currently applied to other proteins. The proteins of the present invention can further be used to generate an antibody which selectively binds the protein.
Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments of these antibodies, and humanized forms.The invention further provides antibodies which selectively bind to one of the proteins of the present invention and hybridomas which produce these antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A.



Law of attraction money in hindi
Project management certification online texas lottery

free online classes johns hopkins



Comments to «Free online classes johns hopkins»

  1. SAXTA_BABA writes:
    Money saving choice and lottery filters the myths and the this world, which.
  2. vitos_512 writes:
    Relation to placing in challenging operate lots.