Invented by Robert D. Fleischmann, Mark D. Adams, Owen White, Hamilton O. Smith, J. Craig Venter, Human Genome Sciences Inc, Johns Hopkins University, Institute for Genomic Research
The Human Genome Sciences Inc, Johns Hopkins University, Institute for Genomic Research invention works as followsThe present invention allows for the complete sequencing of the genome of Haemophilus Influenzae Rd, SEQUID NO:1. The present invention also provides sequence information on computer-readable media and computer-based methods that facilitate its use. The present invention not only provides the complete genome sequence but also identifies more than 1700 fragments of Haemophilus protein coding genes and determines their relative positions to a single Not I restriction enzyme site.
Background for Selected Haemophilus Influene Rd polynucleotides
The present invention is in the field of molecular biology. The present invention discloses compositions containing the nucleotide sequencing of Haemophilus flue, fragments of it and their use in industrial fermentation and pharmaceutical developments.
The complete genome sequence of a living cell organism has not been determined. The first mycobacterium genome sequence is expected to be complete by 1996. E. coli, S. cerevisae and other organisms are also expected to be finished before 1998. The random or directed sequencing of cosmid clones that overlap is being done. “No one has ever tried to randomly determine the sequence of a megabase, or even more than that.
The genes that determine the virulence of H. influenzae have been the focus of interest in medically significant aspects of H. flue biology. The genes that are responsible for the polysaccharide capsular have been sequenced and mapped (Kroll et.al., Mol. Microbiol. 5(6):1549-1560 (1991)). The sequences of several outer membrane proteins (OMPs) have been determined (Langford, et. al. J. Gen. Microbiol. 138:155-159 (1992)). Weiser et. al., J. Bacteriol. 172:3304-3309 (1990)). The need to improve vaccines is a major motivation for the study of outer-membrane components, even though a vaccine was available in 1984. Recent studies have characterized the catalase gene and sequenced it as a potential virulence gene (Bishni, et. al., under review). The H. influenzae gene will help us better understand how H. flues causes infection and how to best combat it.
H. influenzae has a highly effective natural DNA transformation system, which was intensively studied by Kahn and Smith in the serotype d non-encapsulated strain (Kahn & Smith, J. Membrane Biology 81:309-103 (1984). At least 16 transformation-specific genes have been identified and sequenced. Redfield, J. Bacteriol. 173:5612?5618 (1991), and Chandler (Proc. Natl. Acad. Sci. J. Bacteriol. 163(2):629-634 (1985)), and at least seven are directed to the membranes and periplasmic space (Tomb et al., Gene 104:1-10 (1991), Tomb, Proc. 163(2):629-634 ( 1985)). At least seven of these are directed at membranes or periplasmic spaces (Tomb, et al. Gene 104, 1-10 (1991), Tomb, Proc. Natl. Acad. Sci. USA 89:10252-10256 (1992)), where they seem to function either as structural components or for the assembly of DNA transport machinery. H. influenzae transformation has a number interesting features, including sequence-specific uptake, rapid transport of double-stranded donor DNA per cell into a compartment membrane called the transformasome. It also shows linear translocation of one strand of donor DNA to the cytoplasm and synapsis with the chromosome using a single-strand displacement. H. influenzae’s Rd system has been the most extensively studied gram-negative system and is distinct from gram positive systems in many ways.
The H. influenzae Rd genome size has been determined using pulsed-field electrophoresis with restriction digests to be approximately 1.95 Mb. This is approximately 40% of E.coli’s genome (Lee and Smith J. Bacteriol. 170:4402-4405 (1988)). The restriction map for H. influenzae is circular. (Lee and al., J. Bacteriol. 171:3016-3024 (1989) and Redfield and Lee?Haemophilus Influenzae Rd?, pages. 2110-2112, In O’Brien, S. J. (ed), Genetic Maps. Locus Maps for Complex Genomes. Cold Spring Harbor Press. N.Y. Different genes were mapped to restriction fragments using Southern hybridization probing restriction digest DNA bands. This map can be used to verify the assembly of a complete sequence of genomes from randomly sequenced fragments. GenBank currently has about 100 kb non-redundant H. flue DNA sequences. Half of the sequences are from Serotype B and half are Rd.
The present invention is based upon the sequence of the Haemophilus flue Rd genome. The primary nucleotide sequencing of selected ORFs can be found in SEQID NOS:1-10.
The present invention is a method for generating the nucleotide sequencing of the Haemophilus flue Rd genome or a representative portion thereof in a format that can be easily used, analyzed and interpreted by an expert artisan. In one embodiment, the present invention relates to primary sequence information that corresponds to the nucleotide-sequences shown in SEQ ID NoS:1-10.
The present invention also provides nucleotide sequencings that are at least 99.9% similar to the sequence of SEQ ID No:1.
The nucleotide sequencing of SEQID NOS:1-10 or a representative fragment of it, or another nucleotide that is at least 99.9% the same as SEQID NOS:1-10 can be provided on a variety media to make its use easier. In one application, the sequences are recorded on computer-readable media. These media include, but are not limited to, magnetic storage media like floppy disks, CD-ROM, magnetic tape, optical storage media like CD-ROM, electrical storage media (RAM and ROM), and hybrids such as magnetic/optical media.
The invention also provides systems, especially computer-based systems that contain the sequence information described herein stored in a data storage device. These systems can be used to identify commercially relevant fragments of the Haemophilus Influenzae Rd genome.
Another embodiment is directed at isolated fragments from the Haemophilus Influenzae Rd genome. The present invention includes fragments that encode peptides, hereinafter called open reading frames (ORFs), which modulate the expression an operably linked ORF (EMFs), which mediate the uptake a linked DNA fragment in a cell (hereafter UMFs), and fragments that can be used for diagnosing the presence of Haemophilus flue Rd in a sample (hereafter referred to as diagnostic fragments or DFs).
Each of the ORF fragments from the Haemophilus influenzae genome revealed in Table 1(a) and the EMF discovered 5? The ORF can be used as polynucleotide primers in a variety of ways. These sequences are useful as diagnostic probes and diagnostic amplification primers to detect the presence of microbes in a sample. They can also be used for the production commercially valuable pharmaceutical agents or to control gene expression.
The present invention also includes recombinant constructs that contain one or more fragments from the Haemophilus Influenzae Rd genome. The vectors that comprise the recombinant constructs described in the present invention include vectors such as a viral vector or plasmid into which a Haemophilus influenzaeRd fragment has been embedded.
The present invention also provides host cells that contain any fragment of the Haemophilus Influenzae Rd genome. You can choose to use higher eukaryotic hosts such as mammalian cells, lower eukaryotic cells such as yeast cells, or procaryotic cells such as bacteria.
The present invention further relates to the isolation of proteins encoded using the ORFs. Any one of the invention’s proteins can be obtained using any of the many methods known to the art. The simplest form of the amino acid sequence is possible to synthesize using commercially available, peptide synthesizers. Another method is to purify the protein from bacteria cells that naturally produce it. The proteins of the invention can also be extracted from cells that have been modified to express the desired protein.
The invention also provides methods for obtaining homologs to the fragments from the Haemophilus influenzaeRd genome and homologs to the proteins encoded using the ORFs. One skilled in the art can find homologs by using the nucleotide or amino acid sequences described herein as a probe, primers, and techniques like PCR cloning, colony/plaque hybridization and others.
The invention also provides antibodies that selectively bind to one of the proteins described in the invention.” These antibodies can be monoclonal or polyclonal.
The invention also provides hybridomas that produce the above-described antibody. Hybridomas are immortalized cell lines that can secrete a monoclonal antibody.Click here to view the patent on Google Patents.